Compositions, systems, and methods for detecting events using tethers anchored to or adjacent to nanopores

ABSTRACT

Compositions, systems, and methods for detecting events are provided. A composition can include a nanopore including a first side, a second side, and an aperture extending through the first and second sides; and a permanent tether including head and tail regions and an elongated body disposed therebetween. The head region can be anchored to or adjacent to the first or second side of the nanopore. The elongated body including a reporter region can be movable within the aperture responsive to a first event occurring adjacent to the first side of the nanopore. For example, the reporter region is translationally movable toward the first side responsive to the first event, then toward the second side, then toward the first side responsive to a second event. The first event can include adding a first nucleotide to a polynucleotide. The second event can include adding a second nucleotide to the polynucleotide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the following applications, theentire contents of each of which are incorporated by reference herein:

U.S. Provisional Patent Application No. 62/007,248, filed Jun. 3, 2014and entitled “Compositions, Systems, and Methods for Detecting EventsUsing Tethers Anchored to or Adjacent to Nanopores;” and

U.S. Provisional Patent Application No. 62/157,371, filed May 5, 2015and entitled “Compositions, Systems, and Methods for Detecting EventsUsing Tethers Anchored to or Adjacent to Nanopores.”

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 28, 2015, isnamed 12957-178-999_SL.txt and is 2,150 bytes in size.

FIELD

This application generally relates to detecting molecular events, suchas the motion of a molecule or a portion of that molecule.

BACKGROUND

A significant amount of academic and corporate time and energy has beeninvested into detecting events, such as the motion of a molecule or aportion of that molecule, particularly where the molecule is DNA or anenzyme that binds DNA, such as a polymerase. For example, Olsen et al.,“Electronic Measurements of Single-Molecule Processing by DNA PolymeraseI (Klenow Fragment),” JACS 135: 7855-7860 (2013), the entire contents ofwhich are incorporated by reference herein, discloses bioconjugatingsingle molecules of the Klenow fragment (KF) of DNA polymerase I intoelectronic nanocircuits so as to allow electrical recordings ofenzymatic function and dynamic variability with the resolution ofindividual nucleotide incorporation events. Or, for example, Hurt etal., “Specific Nucleotide Binding and Rebinding to Individual DNAPolymerase Complexes Captured on a Nanopore,” JACS 131: 3772-3778(2009), the entire contents of which are incorporated by referenceherein, discloses measuring the dwell time for complexes of DNA with theKF atop a nanopore in an applied electric field. Or, for example, Kim etal., “Detecting single-abasic residues within a DNA strand immobilizedin a biological nanopore using an integrated CMOS sensor,” Sens.Actuators B Chem. 177: 1075-1082 (2012), the entire contents of whichare incorporated by reference herein, discloses using a current orflux-measuring sensor in experiments involving DNA captured in aα-hemolysin nanopore. Or, for example, Garalde et al., “DistinctComplexes of DNA Polymerase I (Klenow Fragment) for Based and SugarDiscrimination during Nucleotide Substrate Selection,” J. Biol. Chem.286: 14480-14492 (2011), the entire contents of which are incorporatedby reference herein, discloses distinguishing KF-DNA complexes on thebasis of their properties when captured in an electric field atop anα-hemolysin pore. Other references that disclose measurements involvingα-hemolysin include the following, all to Howorka et al., the entirecontents of which are incorporated by reference herein: “Kinetics ofduplex formation for individual DNA strands within a single proteinnanopore,” PNAS 98: 12996-13301 (2001); “Probing Distance and ElectricalPotential within a Protein Pore with Tethered DNA,” Biophysical Journal83: 3202-3210 (2002); and “Sequence-specific detection of individual DNAstrands using engineered nanopores,” Nature Biotechnology 19: 636-639(2001).

U.S. Pat. No. 8,652,779 to Turner et al., the entire contents of whichare incorporated by reference herein, discloses compositions and methodsof nucleic acid sequencing using a single polymerase enzyme complexincluding a polymerase enzyme and a template nucleic acid attachedproximal to a nanopore, and nucleotide analogs in solution. Thenucleotide analogs include charge blockade labels that are attached tothe polyphosphate portion of the nucleotide analog such that the chargeblockade labels are cleaved when the nucleotide analog is incorporatedinto a growing nucleic acid. According to Turner, the charge blockadelabel is detected by the nanopore to determine the presence and identityof the incorporated nucleotide and thereby determine the sequence of atemplate nucleic acid. U.S. Patent Publication No. 2014/0051069 toJayasinghe et al., the entire contents of which are incorporated byreference herein, is directed to constructs that include a transmembraneprotein pore subunit and a nucleic acid handling enzyme.

However, previously known compositions, systems, and methods such asdescribed by Olsen, Hurt, Kim, Garalde, Howorka, Turner, and Jayasinghemay not necessarily be sufficiently robust, reproducible, or sensitiveand may not have sufficiently high throughput for practicalimplementation, e.g., demanding commercial applications such as genomesequencing in clinical and other settings that demand cost effective andhighly accurate operation. Accordingly, what is needed are improvedcompositions, systems, and methods for detecting events.

SUMMARY

Embodiments of the present invention provide compositions, systems, andmethods for detecting events using tethers anchored to or adjacent tonanopores.

Under one aspect, a composition includes a nanopore including a firstside, a second side, and an aperture extending through the first andsecond sides; and a permanent tether including a head region, a tailregion, and an elongated body disposed therebetween. The head region canbe anchored to or adjacent to the first side or second side of thenanopore. The elongated body including a reporter region can be movablewithin the aperture responsive to a first event occurring adjacent tothe first side of the nanopore. In one non-limiting example, the headregion can be anchored to a molecule, such as a protein, disposed on thefirst side or second side of the nanopore.

In some embodiments, the reporter region is translationally movablewithin the aperture responsive to the first event. Additionally, oralternatively, the reporter region can be rotationally movable withinthe aperture responsive to the first event. Additionally, oralternatively, the reporter region can be conformationally movablewithin the aperture responsive to the first event.

In some embodiments, the head region is anchored to or adjacent to thefirst side or second side of the nanopore via a covalent bond. The headregion can be anchored to the first side of the nanopore. The tailregion can extend freely toward the second side of the nanopore.

In some embodiments, the reporter region is translationally movabletoward the first side of the nanopore responsive to the first event. Thereporter region can be translationally movable toward the second sideafter the first event. The reporter region further can betranslationally movable toward the first side responsive to a secondevent occurring adjacent to the first side of the nanopore, the secondevent being after the first event. The reporter region further can betranslationally movable toward the second side after the second event.In some embodiments, the first event includes adding a first nucleotideto a polynucleotide. In embodiments that include a second event, thesecond event can include adding a second nucleotide to thepolynucleotide.

An electrical or flux blockade characteristic of the reporter region canbe different than an electrical or flux blockade characteristic ofanother region of the elongated body.

A system can include a composition and measurement circuitry configuredto measure a first current or flux through the aperture or to measure afirst optical signal while the reporter region is moved responsive tothe first event.

In some embodiments, the composition further includes a protein disposedadjacent to the first side of the nanopore, and the first event includesa first conformational change of the protein. The protein is generallynot a native component of a nanopore.

In some embodiments, the head region is anchored to the protein. Thefirst conformational change can move the head region, and the movementof the head region can translationally move the reporter region.

In some embodiments, the protein is in contact with the first side ofthe nanopore. In some embodiments, the protein can be anchored to oradjacent to the first side of the nanopore.

In some embodiments, the protein includes an enzyme. For example, theenzyme can include a polymerase. The first conformational change canoccur responsive to the polymerase acting upon a first nucleotide. Insome embodiments, the first conformational change moves the head region,and the movement of the head region translationally moves the reporterregion. The first nucleotide can be identifiable based on a measuredmagnitude or time duration, or both, of a change in a current or fluxthrough the aperture or a first optical signal responsive to thetranslational movement of the reporter region.

The reporter region further can be translationally movable responsive toa second conformational change of the polymerase occurring responsive tothe polymerase acting upon a second nucleotide. In some embodiments, thefirst nucleotide is identifiable based on a measured magnitude or timeduration, or both, of a first change in a current or flux through theaperture or a first optical signal responsive to the translationalmovement of the reporter region responsive to the first conformationalchange. The second nucleotide can be identifiable based on a measuredmagnitude or time duration, or both, of a second change in the currentor flux through the aperture or a second optical signal responsive tothe translational movement of the reporter region responsive to thesecond conformational change. In some embodiments, the first and secondnucleotides are individually distinguishable from one another based onthe first and second changes in the current or flux or based on thefirst and second optical signals.

In some embodiments, the composition further includes a polymerasedisposed adjacent to the first side of the nanopore, and the first eventincludes the polymerase acting upon a first nucleotide. The firstnucleotide can include an elongated tag including a moiety thatinteracts with the tether. The interaction of the moiety with the tethercan translationally move the reporter region.

In some embodiments, the elongated body of the tether can include asynthetic polymer. In some embodiments, the tether includes a firstoligonucleotide. An abasic nucleotide of the first oligonucleotide candefine the reporter region. Additionally, or alternatively, the moietycan include a second oligonucleotide that hybridizes to the firstoligonucleotide. The hybridization of the second oligonucleotide to thefirst oligonucleotide can shorten the tether by a first amount. In someembodiments, the first nucleotide is identifiable based on a measuredmagnitude or time duration, or both, of change in a current or fluxthrough the aperture or an optical signal responsive to the shorteningof the tether by the first amount. In some embodiments, the reporterregion further is translationally movable toward the first sideresponsive to the polymerase acting upon a second nucleotide. The secondnucleotide can include a third oligonucleotide that hybridizes to thefirst oligonucleotide. The hybridization of the third oligonucleotide tothe first oligonucleotide can shorten the tether by a second amount. Insome embodiments, the first nucleotide is identifiable based on ameasured magnitude or time duration, or both, of a first change in acurrent or flux through the aperture or a first optical signalresponsive to the shortening of the tether by the first amount. Inembodiments that include a second nucleotide, the second nucleotide canbe identifiable based on a measured magnitude or time duration, or both,of a second change in the current or flux through the aperture or asecond optical signal responsive to the shortening of the tether by thesecond amount. In some embodiments, the first and second nucleotides areindividually distinguishable from one another based on the first andsecond changes in the current or flux or based on the first and secondoptical signals.

In some embodiments, the head region is anchored to the first side ofthe nanopore. In some embodiments, the polymerase is in contact with thefirst side of the nanopore. In some embodiments, the polymerase isanchored to or adjacent to the first side of the nanopore.

Some embodiments further include a polymerase disposed on the firstside, the head region being anchored to the polymerase. Some embodimentsfurther include a first nucleotide and first and second polynucleotideseach in contact with the polymerase, the polymerase configured to addthe first nucleotide to the first polynucleotide based on a sequence ofthe second polynucleotide. In some embodiments, the polymerase ismodified so as to delay release of pyrophosphate responsive to additionof the first nucleotide to the first polynucleotide. In someembodiments, the polymerase includes a modified recombinant Φ29, B103,GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7,PR4, PR5, PR722, or L17 polymerase. In some embodiments, the polymeraseincludes a modified recombinant Φ29 DNA polymerase having at least oneamino acid substitution or combination of substitutions selected fromthe group consisting of: an amino acid substitution at position 484, anamino acid substitution at position 198, and an amino acid substitutionat position 381. In some embodiments, the polymerase includes a modifiedrecombinant Φ29 DNA polymerase having at least one amino acidsubstitution or combination of substitutions selected from the groupconsisting of E375Y, K512Y, T368F, A484E, A484Y, N387L, T372Q, T372L,K478Y, 1370W, F198W, and L381A.

In some embodiments, the composition further includes a polymerasedisposed on the first side, the head region being anchored to thepolymerase. Some embodiments further include a first nucleotide andfirst and second polynucleotides each in contact with the polymerase,the polymerase configured to add the first nucleotide to the firstpolynucleotide based on a sequence of the second polynucleotide. In someembodiments, the first nucleotide is coupled to a reversible terminatorthat inhibits the polymerase from adding a second nucleotide to thefirst polynucleotide. In some embodiments, the reversible terminator iscleavable by exposure to light or heat. In some embodiments, thereversible terminator is cleavable by absorption of heat from the light.In some embodiments, the reversible terminator is cleavable by aphotochemical reaction induced by the light. In some embodiments, thereversible terminator is cleavable by reaction with a chemical agent. Insome embodiments, the composition further includes a source of thechemical agent. In some embodiments, the reversible terminator isdisposed on the first side, and the source of the chemical agent isdisposed on the second side such that the chemical agent moves from thesecond side to the first side through the aperture. In some embodiments,the reversible terminator includes azidomethyl (CH₂N₃), and the chemicalagent includes THP.

In some embodiments, an apparatus includes such a composition, whereinthe composition is present in a flow cell and the flow cell isconfigured to replenish reagents that are in contact with thepolymerase.

Under another aspect, a method includes providing a nanopore including afirst side, a second side, and an aperture extending through the firstand second sides; and providing a permanent tether including a headregion, a tail region, and an elongated body disposed therebetween. Thehead region can be anchored to or adjacent to the first or second sideof the nanopore, and the elongated body can include a reporter region.The method can include moving the reporter within the apertureresponsive to a first event occurring adjacent to the first side of thenanopore.

In some embodiments, the reporter region is translationally moved withinthe aperture responsive to the first event. Additionally, oralternatively, the reporter region can be rotationally moved within theaperture responsive to the first event. Additionally, or alternatively,the reporter region is conformationally moved within the apertureresponsive to the first event.

In some embodiments, the head region is anchored to or adjacent to thefirst side or second side of the nanopore via a covalent bond. In someembodiments, the head region is anchored to the first side of thenanopore. In some embodiments, the tail region extends freely toward thesecond side of the nanopore.

In some embodiments, the reporter region is translationally moved towardthe first side of the nanopore responsive to the first event. Someembodiments further include translationally moving the reporter regiontoward the second side after the first event. Some embodiments furtherinclude translationally moving the reporter region toward the first sideresponsive to a second event occurring adjacent to the first side of thenanopore, the second event being after the first event. Some embodimentsfurther include translationally moving the reporter region toward thesecond side after the second event. In some embodiments, the first eventincludes adding a first nucleotide to a polynucleotide. In someembodiments, the second event includes adding a second nucleotide to thepolynucleotide.

In some embodiments, an electrical or flux blockade characteristic ofthe reporter region is different than an electrical or flux blockadecharacteristic of another region of the elongated body.

The method further can include measuring a first current or flux throughthe aperture or a first optical signal while the reporter region ismoved responsive to the first event.

In some embodiments, a protein is disposed adjacent to the first side ofthe nanopore, and the first event includes a first conformational changeof the protein. The head region can be anchored to the protein. Thefirst conformational change can move the head region, and the movementof the head region can translationally move the reporter region.

In some embodiments, the protein is in contact with the first side ofthe nanopore. In some embodiments, the protein is anchored to oradjacent to the first side of the nanopore.

In some embodiments, the protein includes an enzyme. For example, theenzyme can include a polymerase. The first conformational change canoccur responsive to the polymerase acting upon a first nucleotide. Insome embodiments, the first conformational change moves the head region,and the movement of the head region translationally moves the reporterregion. Some embodiments further include identifying the firstnucleotide based on a measured magnitude or time duration, or both, of achange in a current or flux through the aperture or an optical signalresponsive to the translational movement of the reporter region.

Some embodiments further include translationally moving the reporterregion responsive to a second conformational change of the polymeraseoccurring responsive to the polymerase acting upon a second nucleotide.Some embodiments further include identifying the first nucleotide basedon a measured magnitude or time duration, or both, of a first change ina current or flux through the aperture or a first optical signalresponsive to the translational movement of the reporter regionresponsive to the first conformational change. Some embodiments furtherinclude identifying the second nucleotide based on a measured magnitudeor time duration, or both, of a second change in the current or fluxthrough the aperture or a second optical signal responsive to thetranslational movement of the reporter region responsive to the secondconformational change. In some embodiments, the first and secondnucleotides are individually distinguishable from one another based onthe first and second changes in the current or flux or based on thefirst and second optical signals.

Some embodiments include disposing a polymerase adjacent to the firstside of the nanopore, and the first event can include the polymeraseacting upon a first nucleotide. The first nucleotide can include anelongated tag including a moiety that interacts with the tether. Theinteraction of the moiety with the tether can translationally move thereporter region.

In some embodiments, the elongated body of the tether includes asynthetic polymer. In some embodiments, the tether includes a firstoligonucleotide. In some embodiments, an abasic nucleotide of the firstoligonucleotide defines the reporter region. In some embodiments, themoiety includes a second oligonucleotide that hybridizes to the firstoligonucleotide. The hybridization of the second oligonucleotide to thefirst oligonucleotide can shorten the tether by a first amount. Someembodiments further include identifying the first nucleotide based on ameasured magnitude or time duration, or both, of a change in a currentor flux through the aperture or an optical signal responsive to theshortening of the tether by the first amount. Some embodiments alsoinclude translationally moving the reporter region toward the first sideresponsive to the polymerase acting upon a second nucleotide. The secondnucleotide can include a third oligonucleotide that hybridizes to thefirst oligonucleotide. The hybridization of the third oligonucleotide tothe first oligonucleotide can shorten the tether by a second amount.Some embodiments further include identifying the first nucleotide basedon a measured magnitude or time duration, or both, of a first change ina current or flux through the aperture or a first optical signalresponsive to the shortening of the tether by the first amount. Someembodiments also include identifying the second oligonucleotide based ona measured magnitude or time duration, or both, of a second change inthe current or flux through the aperture or a second optical signalresponsive to the shortening of the tether by the second amount. Thefirst and second nucleotides can be individually distinguishable fromone another based on the first and second changes in the current or fluxor based on the first and second optical signals.

In some embodiments, the head region is anchored to the first side ofthe nanopore. In some embodiments, the polymerase is in contact with thefirst side of the nanopore. In some embodiments, the polymerase isanchored to or adjacent to the first side of the nanopore.

In some embodiments, the method includes disposing a polymerase on thefirst side, the head region being anchored to the polymerase. In someembodiments, the method further includes contacting the polymerase witha first nucleotide and with first and second polynucleotides, thepolymerase adding the first nucleotide to the first polynucleotide basedon a sequence of the second polynucleotide. In some embodiments, thepolymerase is modified so as to delay release of pyrophosphateresponsive to addition of the first nucleotide to the firstpolynucleotide. In some embodiments, the polymerase includes a modifiedrecombinant Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1,PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase. In someembodiments, the polymerase includes a modified recombinant Φ29 DNApolymerase having at least one amino acid substitution or combination ofsubstitutions selected from the group consisting of: an amino acidsubstitution at position 484, an amino acid substitution at position198, and an amino acid substitution at position 381. In someembodiments, the polymerase includes a modified recombinant Φ29 DNApolymerase having at least one amino acid substitution or combination ofsubstitutions selected from the group consisting of E375Y, K512Y, T368F,A484E, A484Y, N387L, T372Q, T372L, K478Y, 1370W, F198W, and L381A.

In some embodiments, polymerase is disposed on the first side, the headregion being anchored to the polymerase. In some embodiments, the methodfurther includes contacting the polymerase with a first nucleotide andwith first and second polynucleotides, the polymerase adding the firstnucleotide to the first polynucleotide based on a sequence of the secondpolynucleotide. In some embodiments, the first nucleotide is coupled toa reversible terminator, the method further including inhibiting, by thereversible terminator, the polymerase from adding a second nucleotide tothe first polynucleotide. In some embodiments, the method furtherincludes cleaving the reversible terminator by exposure to light orheat. Some embodiments include cleaving the reversible terminator byabsorption of heat from the light. Some embodiments include cleaving thereversible terminator by a photochemical reaction induced by the light.Some embodiments include cleaving the reversible terminator by reactionwith a chemical agent. Some embodiments include providing a source ofthe chemical agent. Some embodiments include flowing fluid past thepolymerase to remove the chemical agent. Some embodiments includesupplying new reagents to the polymerase by fluid flow. In someembodiments, the reversible terminator is disposed on the first side andthe source of the chemical agent is disposed on the second side, themethod including moving the chemical agent from the second side to thefirst side through the aperture. In some embodiments, the reversibleterminator includes azidomethyl (CH₂N₃), and the chemical agent includesTHP.

Under yet another aspect, a composition includes a nanopore including afirst side, a second side, and an aperture extending through the firstand second sides; and a permanent tether including a head region, a tailregion, and an elongated body disposed therebetween. The head region canbe anchored to or adjacent to the first side or second side of thenanopore, and the elongated body can include a moiety. A polymerase canbe disposed adjacent to the first side of the nanopore. The compositionalso can include a first nucleotide including a first elongated tag. Thefirst elongated tag can include a first moiety that interacts with themoiety of the tether responsive to the polymerase acting upon the firstnucleotide.

In some embodiments, the head region is anchored to or adjacent to thefirst side or second side of the nanopore via a covalent bond. Forexample, in some embodiments, the head region is anchored to the firstside of the nanopore. In some embodiments, the tail region extendsfreely toward the second side of the nanopore. In some embodiments, thetail region is movable between the first and second side of the nanoporeresponsive to an applied voltage. Or, for example, in some embodiments,the head region is anchored to the second side of the nanopore. In someembodiments, the tail region extends freely toward the first side of thenanopore. In some embodiments, the tail region is movable between thefirst and second side of the nanopore responsive to an applied voltage.

The polymerase can be in contact with the first side of the nanopore.The polymerase can be anchored to or adjacent to the first side of thenanopore.

In some embodiments, the interaction between the first moiety and themoiety of the tether defines a duplex. The nanopore further can includea constriction disposed between the first and second sides. Theanchoring of the head region to or adjacent to the first or second sideof the nanopore, or to the polymerase, can inhibit movement of theduplex through the constriction. Alternatively, or additionally, theduplex can be sufficiently large as to inhibit movement of the duplexthrough the constriction.

In some embodiments, the first elongated tag of the first nucleotidefurther includes a first reporter region. Optionally, the first reporterregion can be configured to be disposed within the aperture responsiveto the first moiety interacting with the moiety of the tether. A systemcan include any such composition and measurement circuitry configured tomeasure a current or flux through the aperture or an optical signalwhile the first reporter region is disposed within the aperture. Thecurrent or flux or optical signal can be based on an electrical or fluxblockade characteristic of the first reporter region, and the firstnucleotide can be identifiable based on the current or flux or opticalsignal.

In some embodiments, a composition further includes a second nucleotideincluding a second elongated tag, the second elongated tag including asecond moiety that interacts with the moiety of the tether responsive tothe polymerase acting upon the second nucleotide. The second elongatedtag further can include a second reporter region. In some embodiments,the second reporter region is configured to be disposed within theaperture responsive to the second moiety interacting with the moiety ofthe tether. A system can include any such composition and measurementcircuitry configured to measure a first current or flux through theaperture or a first optical signal while the first reporter region isdisposed within the aperture and a second current or flux through theaperture or a second optical signal while the second reporter region isdisposed within the aperture. The first current or flux or the firstoptical signal can be based on a first electrical or flux blockadecharacteristic of the first reporter region. The first nucleotide can beidentifiable based on the first current or flux or the first opticalsignal. The second current or flux or the second optical signal can bebased on a second electrical or flux blockade characteristic of thesecond reporter region. The second nucleotide can be identifiable basedon the second current or flux or the second optical signal. The firstand second nucleotides can be individually distinguishable from oneanother based on the first and second currents or fluxes or the firstand second optical signals.

In some embodiments of the present compositions, the first elongated tagis cleavable from the first nucleotide responsive to the polymeraseacting upon the first nucleotide, and the second elongated tag iscleavable from the second nucleotide responsive to the polymerase actingupon the second nucleotide.

In some embodiments, the elongated body of the tether includes asynthetic polymer. In some embodiments, the moiety of the tetherincludes a first oligonucleotide. In some embodiments, the first moietyincludes a second oligonucleotide that hybridizes to the firstoligonucleotide. An abasic nucleotide of the second oligonucleotide candefine the reporter region. In some embodiments, the second moietyincludes a third oligonucleotide that hybridizes to the firstoligonucleotide. In some embodiments, the first moiety and the secondmoiety are the same as one another.

In some embodiments, the elongated body of the tether further includes areporter region. The reporter region can be disposed at a predefinedlocation relative to the first moiety responsive to the interaction ofthe first moiety with the moiety of the tether. The reporter region canbe translationally movable within the aperture responsive to a firstapplied voltage. In some embodiments, the nanopore further including aconstriction disposed between the first and second sides. The reporterregion can be translationally movable to a first predetermined locationrelative to the constriction responsive to the first applied voltage.

An electrical or flux blockade characteristic of the reporter region canbe different than an electrical or flux blockade characteristic ofanother region of the elongated body.

A system can include such a composition and measurement circuitryconfigured to measure a current or flux through the aperture or anoptical signal while the reporter region is disposed at the firstpredetermined location. The current or flux or optical signal can bebased on the electrical or flux blockade characteristic of the reporterregion and the first predetermined location of the reporter region, andthe first nucleotide can be identifiable based on the current or flux oroptical signal.

In some embodiments, the first moiety and the moiety of the tether aredissociable responsive to the first applied voltage. The moiety of thetether can be translationally movable through the constrictionresponsive to dissociation of the first moiety and the moiety of thetether. In some embodiments, the first moiety interacts with the moietyof the tether responsive to a second applied voltage subsequent to thefirst applied voltage. In some embodiments, the composition furtherincludes a second nucleotide including a second elongated tag, thesecond elongated tag including a second moiety that interacts with themoiety of the tether responsive to the polymerase acting upon the secondnucleotide. The reporter region can be disposed at a predeterminedlocation relative to the second moiety responsive to the interaction ofthe second moiety with the moiety of the tether. In some embodiments,the reporter region is translationally movable within the apertureresponsive to a second applied voltage. In some embodiments, thenanopore further includes a constriction disposed between the first andsecond sides. The reporter region can be translationally movable to asecond location relative to the constriction responsive to the secondapplied voltage.

An electrical or flux blockade characteristic of the reporter region canbe different than an electrical or flux blockade characteristic ofanother region of the elongated body.

A system can include such a composition and measurement circuitryconfigured to measure a first current or flux through the aperture or afirst optical signal while the reporter region is disposed at the firstlocation responsive to the first applied voltage, and to measure asecond current or flux through the aperture or a second optical signalwhile the reporter region is disposed at the second location responsiveto the second applied voltage. The first current or flux or firstoptical signal can be based on the electrical or flux blockadecharacteristic of the reporter region and the first predeterminedlocation of the reporter region. The first nucleotide can beidentifiable based on the first current or flux or first optical signal.The second current or flux or second optical signal can be based on theelectrical or flux blockade characteristic of the reporter region andthe second predetermined location of the reporter region. The secondnucleotide can be identifiable based on the second current or flux orsecond optical signal. In some embodiments, the first and secondnucleotides are individually distinguishable from one another based onthe first current or flux and the second current or flux or based on thefirst and second optical signals. In some embodiments, the first andsecond voltages have the same magnitude as one another, and the secondvoltage is subsequent to the first voltage.

In some embodiments of the present compositions, the first elongated tagis cleavable from the first nucleotide responsive to the polymeraseacting upon the first nucleotide, and the second elongated tag iscleavable from the second nucleotide responsive to the polymerase actingupon the second nucleotide.

In some embodiments, the elongated body of the tether includes asynthetic polymer. In some embodiments, the moiety of the tetherincludes a first oligonucleotide. An abasic nucleotide of the firstoligonucleotide can define the reporter region. The first moiety caninclude a second oligonucleotide that hybridizes to the firstoligonucleotide. The second moiety can include a third oligonucleotidethat hybridizes to the first oligonucleotide. The first moiety and thesecond moiety can be different than one another. The reporter region canbe translationally movable within the aperture responsive to thepolymerase acting upon the first nucleotide. Alternatively, oradditionally, the reporter region can be rotationally movable within theaperture responsive to the polymerase acting upon the first nucleotide.Alternatively, or additionally, the reporter region can beconformationally movable within the aperture responsive to thepolymerase acting upon the first nucleotide.

In some embodiments, the elongated body of the tether includes asynthetic polymer. In some embodiments, the moiety of the tetherincludes a first oligonucleotide. An abasic nucleotide of the firstoligonucleotide can define the reporter region. The first moiety caninclude a second oligonucleotide that hybridizes to the firstoligonucleotide. The hybridization of the second oligonucleotide to thefirst oligonucleotide can shorten the tether by a first amount. Thefirst nucleotide can be identifiable based on a measured magnitude ortime duration, or both, of change in a current or flux through theaperture or an optical signal responsive to the shortening of the tetherby the first amount.

In some embodiments, the first elongated tag of the first nucleotidefurther includes a first fluorescent resonant energy transfer (FRET)pair partner, and the tether further includes a second FRET pairpartner. The first FRET pair partner and the second FRET pair partnercan interact with one another responsive to the polymerase acting uponthe first nucleotide. A first wavelength emitted responsive to theinteraction between the first FRET pair partner and the second FRET pairpartner is detectable. The composition further can include a secondnucleotide including a second elongated tag, the second elongated tagincluding a third fluorescent resonant energy transfer (FRET) pairpartner. A system can include such a composition. The third FRET pairpartner and the second FRET pair partner can interact with one anotherresponsive to the polymerase acting upon the second nucleotide. Thesystem can include an optical detection system configured to detect asecond wavelength emitted responsive to the interaction between thethird FRET pair partner and the second FRET pair partner. The first andsecond nucleotides can be individually distinguishable from one anotherbased on the first and second wavelengths.

In some embodiments, the composition further includes first and secondpolynucleotides in contact with the polymerase, the polymeraseconfigured to add the first nucleotide to the first polynucleotide basedon a sequence of the second polynucleotide. In some embodiments, thepolymerase is modified so as to delay release of pyrophosphateresponsive to addition of the first nucleotide to the firstpolynucleotide. In some embodiments, the polymerase includes a modifiedrecombinant Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1,PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase. In someembodiments, the polymerase includes a modified recombinant Φ29 DNApolymerase having at least one amino acid substitution or combination ofsubstitutions selected from the group consisting of: an amino acidsubstitution at position 484, an amino acid substitution at position198, and an amino acid substitution at position 381. In someembodiments, the polymerase includes a modified recombinant Φ29 DNApolymerase having at least one amino acid substitution or combination ofsubstitutions selected from the group consisting of E375Y, K512Y, T368F,A484E, A484Y, N387L, T372Q, T372L, K478Y, 1370W, F198W, and L381A.

In some embodiments, the first moiety and the moiety of the tether areconfigured to hybridize with one another so as to form a hairpinstructure. A system can include such a composition and a voltage sourceconfigured to apply a voltage across the first and second sides. Thefirst moiety and the moiety of the tether can be configured todehybridize from one another responsive to the voltage in a two-stepprocess.

In some embodiments, the first elongated tag further includes a secondmoiety, the composition further including a third moiety anchored to oradjacent to the first side or second side of the nanopore, the secondmoiety and the third moiety interacting responsive to addition of thefirst nucleotide to the first polynucleotide. A system can include sucha composition and a voltage source configured to apply a voltage acrossthe first and second sides. In some embodiments, the first moiety andthe moiety of the tether are configured to separate from one anotherresponsive to the voltage in a first process, and the second moiety andthe third moiety are configured to separate from one another responsiveto the voltage in a second process.

In some embodiments, the composition further includes first and secondpolynucleotides in contact with the polymerase, the polymeraseconfigured to add the first nucleotide to the first polynucleotide basedon a sequence of the second polynucleotide. In some embodiments, thefirst elongated tag further includes a reversible terminator thatinhibits the polymerase from adding a second nucleotide to the firstpolynucleotide. In some embodiments, the reversible terminator iscleavable by exposure to light or heat. In some embodiments, thereversible terminator is cleavable by absorption of heat from the light.In some embodiments, the reversible terminator is cleavable by aphotochemical reaction induced by the light. In some embodiments, thereversible terminator is cleavable by reaction with a chemical agent.Some embodiments further include a source of the chemical agent. In someembodiments, the reversible terminator is disposed on the first side,and the source of the chemical agent is disposed on the second side suchthat the chemical agent moves from the second side to the first sidethrough the aperture. In some embodiments, the reversible terminatorincludes azidomethyl (CH₂N₃), and the chemical agent includes THP.

In some embodiments, an apparatus includes such a composition, whereinthe composition is present in a flow cell and the flow cell isconfigured to replenish reagents that are in contact with thepolymerase.

Under still another aspect, a method includes providing a nanoporeincluding a first side, a second side, and an aperture extending throughthe first and second sides; and providing a permanent tether including ahead region, a tail region, and an elongated body disposed therebetween.The head region can be anchored to or adjacent to the first side orsecond side of the nanopore, and the elongated body can include amoiety. The method further can include providing a polymerase disposedadjacent to the first side of the nanopore, and providing a firstnucleotide including a first elongated tag, the first elongated tagincluding a moiety. The method further can include acting upon the firstnucleotide with the polymerase; and interacting the first moiety withthe moiety of the tether responsive to the polymerase acting upon thefirst nucleotide.

In some embodiments, the head region is anchored to or adjacent to thefirst side or second side of the nanopore via a covalent bond. Forexample, in some embodiments, the head region is anchored to the firstside of the nanopore. In some embodiments, the tail region extendsfreely toward the second side of the nanopore. Some embodiments includemoving the tail region between the first and second side of the nanoporeresponsive to an applied voltage. Or, for example, in some embodiments,the head region is anchored to the second side of the nanopore. In someembodiments, the tail region extends freely toward the first side of thenanopore. Some embodiments include moving the tail region between thefirst and second side of the nanopore responsive to an applied voltage.

In some embodiments, the polymerase is in contact with the first side ofthe nanopore. In some embodiments, the polymerase is anchored to oradjacent to the first side of the nanopore.

In some embodiments, the interaction between the first moiety and themoiety of the tether defines a duplex. The nanopore further can includea constriction disposed between the first and second sides. The methodfurther can include inhibiting movement of the duplex through theconstriction via the anchoring of the head region to or adjacent to thefirst or second side of the nanopore. Additionally, or alternatively,the duplex can be sufficiently large as to inhibit movement of theduplex through the constriction.

In some embodiments, the first elongated tag of the first nucleotidefurther includes a first reporter region. Some embodiments includedisposing the first reporter region within the aperture responsive tothe first moiety interacting with the moiety of the tether. Someembodiments further include measuring a current or flux through theaperture or an optical signal while the first reporter region isdisposed within the aperture. In some embodiments, the current or fluxor optical signal is based on an electrical or flux blockadecharacteristic of the first reporter region, and the first nucleotide isidentifiable based on the current or flux or optical signal. Someembodiments further include providing a second nucleotide including asecond elongated tag, the second elongated tag including a secondmoiety, acting upon the second nucleotide with the polymerase, andinteracting the second moiety with the moiety of the tether responsiveto the polymerase acting upon the second nucleotide. The secondelongated tag further can include a second reporter region. Someembodiments include disposing the second reporter region within theaperture responsive to the second moiety interacting with the moiety ofthe tether. Some embodiments include measuring a first current or fluxthrough the aperture or a first optical signal while the first reporterregion is disposed within the aperture and measuring a second current orflux through the aperture or a second optical signal while the secondreporter region is disposed within the aperture. In some embodiments,the first current or flux or the first optical signal is based on afirst electrical or flux blockade characteristic of the first reporterregion, the first nucleotide is identifiable based on the first currentor flux or first optical signal, the second current or flux or thesecond optical signal is based on a second electrical or flux blockadecharacteristic of the second reporter region, and the second nucleotideis identifiable based on the second current or flux or second opticalsignal. In some embodiments, the first and second nucleotides areindividually distinguishable from one another based on the first currentor flux and second current or flux or first and second optical signals.Some embodiments further include cleaving the first elongated tag fromthe first nucleotide responsive to the polymerase acting upon the firstnucleotide, and cleaving the second elongated tag from the secondnucleotide responsive to the polymerase acting upon the secondnucleotide.

In some embodiments, the elongated body of the tether includes asynthetic polymer. In some embodiments, the moiety of the tetherincludes a first oligonucleotide. In some embodiments, the first moietyincludes a second oligonucleotide that hybridizes to the firstoligonucleotide. In some embodiments, an abasic nucleotide of the secondoligonucleotide defines the reporter region. In some embodiments, thesecond moiety includes a third oligonucleotide that hybridizes to thefirst oligonucleotide. In some embodiments, the first moiety and thesecond moiety are the same as one another. In some embodiments, theelongated body of the tether further includes a reporter region. Someembodiments further include disposing the reporter region at apredefined location relative to the first moiety responsive to theinteraction of the first moiety with the moiety of the tether. Someembodiments further include translationally moving the reporter regionwithin the aperture responsive to a first applied voltage. The nanoporefurther can include a constriction disposed between the first and secondsides. The reporter region can be translationally moved to a firstpredetermined location relative to the constriction responsive to thefirst applied voltage. An electrical or flux blockade characteristic ofthe reporter region can be different than an electrical or flux blockadecharacteristic of another region of the elongated body. Some embodimentsfurther include measuring a current or flux through the aperture or anoptical signal while the reporter region is disposed at the firstpredetermined location. In some embodiments, the current or flux oroptical signal is based on the electrical or flux blockadecharacteristic of the reporter region and the first predeterminedlocation of the reporter region, and the first nucleotide isidentifiable based on the current or flux or optical signal. Someembodiments further include dissociating the first moiety and the moietyof the tether responsive to the first applied voltage. Some embodimentsinclude translationally moving the moiety of the tether through theconstriction responsive to dissociation of the first moiety and themoiety of the tether. Some embodiments include interacting the firstmoiety with the moiety of the tether responsive to a second appliedvoltage subsequent to the first applied voltage.

In some embodiments, the method further includes providing a secondnucleotide including a second elongated tag, the second elongated tagincluding a second moiety that interacts with the moiety of the tetherresponsive to the polymerase acting upon the second nucleotide. Themethod can include disposing the reporter region at a predeterminedlocation relative to the second moiety responsive to the interaction ofthe second moiety with the moiety of the tether. The method can includetranslationally moving the reporter region within the apertureresponsive to a second applied voltage. The nanopore further can includea constriction disposed between the first and second sides. The reporterregion can be translationally moved to a second location relative to theconstriction responsive to the second applied voltage. An electrical orflux blockade characteristic of the reporter region can be differentthan an electrical or flux blockade characteristic of another region ofthe elongated body. Some embodiments further include measuring a firstcurrent or flux through the aperture or a first optical signal while thereporter region is disposed at the first location responsive to thefirst applied voltage, and measuring a second current or flux or asecond optical signal through the aperture while the reporter region isdisposed at the second location responsive to the second appliedvoltage. In some embodiments, the first current or flux or first opticalsignal is based on the electrical or flux blockade characteristic of thereporter region and the first predetermined location of the reporterregion. The first nucleotide can be identifiable based on the firstcurrent or flux or first optical signal. The second current or flux orsecond optical signal can be based on the electrical or flux blockadecharacteristic of the reporter region and the second predeterminedlocation of the reporter region. The second nucleotide can beidentifiable based on the second current or flux or second opticalsignal. In some embodiments, the first and second nucleotides areindividually distinguishable from one another based on the first currentor flux and second current or flux or based on the first and secondoptical signals. The first and second voltages can have the samemagnitude as one another, and the second voltage can be subsequent tothe first voltage. In some embodiments, the first elongated tag iscleavable from the first nucleotide responsive to the polymerase actingupon the first nucleotide, and the second elongated tag is cleavablefrom the second nucleotide responsive to the polymerase acting upon thesecond nucleotide.

In some embodiments, the elongated body of the tether includes asynthetic polymer. In some embodiments, the moiety of the tetherincludes a first oligonucleotide. An abasic nucleotide of the firstoligonucleotide can define the reporter region. The first moiety caninclude a second oligonucleotide that hybridizes to the firstoligonucleotide. The second moiety can include a third oligonucleotidethat hybridizes to the first oligonucleotide. The first moiety and thesecond moiety can be different than one another.

In some embodiments, the reporter region is translationally movablewithin the aperture responsive to the polymerase acting upon the firstnucleotide. Additionally, or alternatively, the reporter region can berotationally movable within the aperture responsive to the polymeraseacting upon the first nucleotide. Additionally, or alternatively, thereporter region can be conformationally movable within the apertureresponsive to the polymerase acting upon the first nucleotide.

The elongated body of the tether can include a synthetic polymer. Themoiety of the tether can include a first oligonucleotide. An abasicnucleotide of the first oligonucleotide can define the reporter region.The first moiety can include a second oligonucleotide that hybridizes tothe first oligonucleotide. The hybridization of the secondoligonucleotide to the first oligonucleotide can shorten the tether by afirst amount. The first nucleotide can be identifiable based on ameasured magnitude or time duration, or both, of change in a current orflux through the aperture or an optical signal responsive to theshortening of the tether by the first amount.

In some embodiments, the first elongated tag of the first nucleotidefurther includes a first fluorescent resonant energy transfer (FRET)pair partner, and the tether further includes a second FRET pairpartner. The first FRET pair partner and the second FRET pair partnercan interact with one another responsive to the polymerase acting uponthe first nucleotide. The method further can include detecting a firstwavelength emitted responsive to the interaction between the first FRETpair partner and the second FRET pair partner. The method further caninclude providing a second nucleotide including a second elongated tag,the second elongated tag including a third fluorescent resonant energytransfer (FRET) pair partner. The third FRET pair partner and the secondFRET pair partner can interact with one another responsive to thepolymerase acting upon the second nucleotide. The method further caninclude detecting a second wavelength emitted responsive to theinteraction between the third FRET pair partner and the second FRET pairpartner. The first and second nucleotides can be individuallydistinguishable from one another based on the first and secondwavelengths.

In some embodiments, the method further includes disposing a polymeraseon the first side, the head region being anchored to the polymerase. Insome embodiments, the method further includes contacting the polymerasewith a first nucleotide and with first and second polynucleotides, thepolymerase adding the first nucleotide to the first polynucleotide basedon a sequence of the second polynucleotide. In some embodiments, thepolymerase is modified so as to delay release of pyrophosphateresponsive to addition of the first nucleotide to the firstpolynucleotide. In some embodiments, the polymerase includes a modifiedrecombinant Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1,PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase. In someembodiments, the polymerase includes a modified recombinant Φ29 DNApolymerase having at least one amino acid substitution or combination ofsubstitutions selected from the group consisting of: an amino acidsubstitution at position 484, an amino acid substitution at position198, and an amino acid substitution at position 381. In someembodiments, the polymerase includes a modified recombinant Φ29 DNApolymerase having at least one amino acid substitution or combination ofsubstitutions selected from the group consisting of E375Y, K512Y, T368F,A484E, A484Y, N387L, T372Q, T372L, K478Y, 1370W, F198W, and L381A.

In some embodiments, the first moiety and the moiety of the tetherhybridize with one another so as to form a hairpin structure. In someembodiments, the method further includes applying a voltage across thefirst and second sides. In some embodiments, the first moiety and themoiety of the tether dehybridize from one another responsive to thevoltage in a two-step process.

In some embodiments, the first elongated tag further includes a secondmoiety, a third moiety anchored to or adjacent to the first side orsecond side of the nanopore, the second moiety and the third moietyinteracting responsive to addition of the first nucleotide to the firstpolynucleotide. In some embodiments, the method further includesapplying a voltage across the first and second sides. In someembodiments, the first moiety and the moiety of the tether separate fromone another responsive to the voltage in a first process, and the secondmoiety and the third moiety separate from one another responsive to thevoltage in a second process.

In some embodiments, the method includes disposing a polymerase on thefirst side, the head region being anchored to the polymerase. Someembodiments include contacting the polymerase with a first nucleotideand with first and second polynucleotides, the polymerase adding thefirst nucleotide to the first polynucleotide based on a sequence of thesecond polynucleotide. In some embodiments, the first elongated tagincludes a reversible terminator, the method further includinginhibiting, by the reversible terminator, the polymerase from adding asecond nucleotide to the first polynucleotide. Some embodiments includecleaving the reversible terminator by exposure to light or heat. Someembodiments include cleaving the reversible terminator by absorption ofheat from the light. Some embodiments include cleaving the reversibleterminator by a photochemical reaction induced by the light. Someembodiments include cleaving the reversible terminator by reaction witha chemical agent. Some embodiments include providing a source of thechemical agent. In some embodiments, the reversible terminator isdisposed on the first side and the source of the chemical agent isdisposed on the second side, the method including moving the chemicalagent from the second side to the first side through the aperture. Insome embodiments, the reversible terminator includes azidomethyl(CH₂N₃), and the chemical agent includes THP.

Some embodiments include flowing fluid past the polymerase to remove thechemical agent. Some embodiments further include supplying new reagentsto the polymerase by fluid flow.

Under another aspect, a composition includes a nanopore including afirst side, a second side, and an aperture extending through the firstand second sides. The composition also can include a permanent tetherincluding a head region, a tail region, and an elongated body disposedtherebetween, the head region being anchored to a polymerase, theelongated body including a moiety. The polymerase can be disposedadjacent to the first side of the nanopore. The composition also caninclude a first nucleotide including a first elongated tag, the firstelongated tag including a first moiety that interacts with the moiety ofthe tether responsive to the polymerase acting upon the firstnucleotide.

In some embodiments, the tail region includes a first nucleic acid. Someembodiments further include a second nucleic acid to which the firstnucleic acid is hybridized. In some embodiments, the head region isdisposed on the first side of the nanopore, and the tail region isdisposed on the second side. In some embodiments, the head region isanchored to the polymerase. In some embodiments, the interaction betweenthe first nucleic acid and the second nucleic acid defines a duplex. Insome embodiments, the nanopore further includes a constriction disposedbetween the first and second sides. In some embodiments, the duplex issufficiently large as to inhibit movement of the duplex through theconstriction. In some embodiments, the tether including the duplexinhibits separation of the polymerase from the nanopore.

Under another aspect, a system includes such a composition andmeasurement circuitry configured to measure a current or flux throughthe constriction or an optical signal. In some embodiments, the currentor flux or optical signal is based on the first moiety, and the firstnucleotide is identifiable based on the current or flux or opticalsignal. In some embodiments, the first elongated tag or the elongatedbody further includes a reporter region, the current or flux or opticalsignal is based on the reporter region being disposed within theaperture, and the first nucleotide is identifiable based on the currentor flux or optical signal.

Under another aspect, a method includes providing a nanopore including afirst side, a second side, and an aperture extending through the firstand second sides. The method also can include providing a permanenttether including a head region, a tail region, and an elongated bodydisposed therebetween, the head region being anchored to a polymerase,the elongated body including a moiety. The method also can includeproviding the polymerase disposed adjacent to the first side of thenanopore. The method also can include providing a first nucleotideincluding a first elongated tag, the first elongated tag including amoiety. The method also can include acting upon the first nucleotidewith the polymerase. The method also can include interacting the firstmoiety with the moiety of the tether responsive to the polymerase actingupon the first nucleotide.

In some embodiments, the tail region includes a first nucleic acid. Someembodiments further include hybridizing a second nucleic acid to thefirst nucleic acid. Some embodiments further include disposing the headregion on the first side of the nanopore and disposing the tail regionon the second side. Some embodiments further include anchoring the headregion to the polymerase. In some embodiments, the interaction betweenthe first nucleic acid and the second nucleic acid defines a duplex. Insome embodiments, the nanopore further includes a constriction disposedbetween the first and second sides. Some embodiments further includeinhibiting, by a size of the duplex, movement of the duplex through theconstriction. Some embodiments further include including inhibiting, bythe tether including the duplex, separation of the polymerase from thenanopore. Some embodiments further include measuring a current or fluxthrough the constriction or an optical signal. In some embodiments, thecurrent or flux or optical signal is based on the first moiety, themethod further including identifying the first nucleotide based on thecurrent or flux or optical signal. In some embodiments, the firstelongated tag or the elongated body further includes a reporter region,wherein the current or flux or optical signal is based on the reporterregion being disposed within the aperture, the method further includingidentifying the first nucleotide based on the current or flux or opticalsignal.

Under another aspect, a method of making a nanopore sequencing deviceincludes providing a chamber including a first liquid medium separatedfrom a second liquid medium by a nanopore, the nanopore including afirst side in contact with the first liquid medium, a second side incontact with the second liquid medium, and an aperture extending throughthe first and second sides. The method also can include providing apolymerase to the first liquid medium, wherein the polymerase includes atether, the tether including a head region, a tail region, and anelongated body disposed therebetween, the head region being anchored tothe polymerase. The method also can include providing a capture moietyto the second liquid medium. The method also can include applying acurrent or flux through the nanopore to translocate the tail region ofthe tether through the nanopore. The method also can include binding thecapture moiety to the tail region of the tether, thereby retaining thetether in the nanopore.

In some embodiments, the tether includes a nucleic acid. In someembodiments, the tail region includes a nucleic acid. In someembodiments, the capture moiety includes a nucleic acid that iscomplementary to the nucleic acid of the tail region. In someembodiments, the capture moiety binds covalently to the tail region. Insome embodiments, the capture moiety binds non-covalently to the tailregion.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1M schematically illustrate compositions including variousconfigurations of tethers anchored to or adjacent to nanopores,according to some embodiments of the present invention.

FIG. 2A schematically illustrates a system including measurementcircuitry configured to measure movement of a reporter region within theaperture of a nanopore, according to some embodiments of the presentinvention.

FIG. 2B is a plot of an exemplary signal that can be generated duringuse of the system of FIG. 2A, according to some embodiments of thepresent invention.

FIG. 2C schematically illustrates a plan view of a system includingmeasurement circuitry configured to measure movement of respectivereporter regions within the respective apertures of an array ofnanopores, according to some embodiments of the present invention.

FIG. 3A illustrates a method for detecting an event using a compositionincluding a tether anchored to or adjacent to a nanopore, according tosome embodiments of the present invention.

FIG. 3B illustrates a method for preparing a composition including atether and a polymerase adjacent to a nanopore, according to someembodiments of the present invention.

FIG. 4A illustrates a method for detecting a conformational change of amolecule using a composition including a tether anchored to or adjacentto a nanopore, according to some embodiments of the present invention.

FIG. 4B illustrates a method for detecting action of a polymerase upon anucleotide using a composition including a tether anchored to oradjacent to a nanopore, according to some embodiments of the presentinvention.

FIGS. 5A-5B schematically illustrate a composition including a tetheranchored adjacent to a nanopore and configured for use in detecting aconformational change of a molecule disposed adjacent to the nanopore,according to some embodiments of the present invention.

FIGS. 6A-6B schematically illustrate exemplary conformational changes ofa polymerase.

FIGS. 6C-6D schematically illustrate a composition including a tetheranchored to a polymerase disposed adjacent to a nanopore and configuredfor use in detecting a conformational change of the polymeraseresponsive to action of the polymerase upon a nucleotide, according tosome embodiments of the present invention.

FIGS. 7A-7B schematically illustrate a composition including a tetheranchored to or adjacent to a nanopore and configured for use indetecting action of a protein upon a nucleotide, according to someembodiments of the present invention.

FIG. 8A schematically illustrates a composition including a tetheranchored to a nanopore and configured for use in detecting action of apolymerase upon a nucleotide, according to some embodiments of thepresent invention.

FIG. 8B schematically illustrates an exemplary nucleotide including anelongated tag including a moiety that interacts with the tether of FIG.8A during use in detecting action of a polymerase upon the nucleotide,according to some embodiments of the present invention.

FIGS. 9A-9B schematically illustrate movement of an exemplary tetherresponsive to hybridization with a moiety of an elongated tag of anexemplary nucleotide during use in detecting action of a polymerase uponthe nucleotide, according to some embodiments of the present invention.

FIGS. 10A-10B schematically illustrate exemplary nucleotides includingelongated tags including respective moieties that can interact with anexemplary tether during use in detecting action of a polymerase upon thenucleotides, according to some embodiments of the present invention.

FIG. 10C schematically illustrates an exemplary tether and moieties thatcan interact with the tether during use in detecting action of apolymerase upon a nucleotide, according to some embodiments of thepresent invention. Sequence disclosed as SEQ ID NO: 5.

FIGS. 11A-11D illustrate exemplary calculations of interactions betweena tether and moieties, according to some embodiments of the presentinvention. Sequence disclosed as SEQ ID NO: 5.

FIG. 12A illustrates a model that can be used to calculate interactionsbetween a tether and moieties, according to some embodiments of thepresent invention.

FIG. 12B illustrates an exemplary calculation of an interaction betweena tether and a moiety, according to some embodiments of the presentinvention. “Loop” disclosed as SEQ ID NO:6 and “Full sequence” disclosedas SEQ ID NO:7.

FIG. 12C illustrates a stable structure calculated based on the model ofFIG. 12A.

FIGS. 13A-13E schematically illustrate interactions between an exemplarytether and moieties of respective nucleotides, according to someembodiments of the present invention. Sequences disclosed as SEQ IDNO:5.

FIG. 14 is a plot of an exemplary signal that can be generated duringinteractions such as illustrated FIGS. 13A-13E, according to someembodiments of the present invention.

FIG. 15 illustrates an alternative method for detecting action of apolymerase upon a nucleotide using a composition including a tetheranchored to or adjacent to a nanopore, according to some embodiments ofthe present invention.

FIG. 16 schematically illustrates an alternative composition including atether anchored to or adjacent to a nanopore and configured for use indetecting action of a polymerase upon a nucleotide, according to someembodiments of the present invention.

FIGS. 17A-17B schematically illustrate a composition including a tetheranchored to or adjacent to a nanopore and configured for use indetecting action of a polymerase upon a nucleotide that includes anelongated tag including a reporter region, according to some embodimentsof the present invention.

FIGS. 18A-18D schematically illustrate a composition including a tetheranchored to or adjacent to a nanopore and configured for use indetecting action of a polymerase upon a nucleotide using a change inelectrical or flux blockade potential across the nanopore, according tosome embodiments of the present invention.

FIG. 18E illustrates an exemplary signal that can be generated duringuse of a composition such as illustrated in FIGS. 18A-18D, according tosome embodiments of the present invention.

FIGS. 19A-19B schematically illustrate a composition including a tetheranchored to or adjacent to a nanopore and configured for use indetecting action of a polymerase upon a nucleotide that includes anelongated tag including a reporter region, according to some embodimentsof the present invention.

FIG. 19C schematically illustrate exemplary nucleotides includingelongated tags including respective reporter regions and moieties thatcan bond to an exemplary tether during use in detecting action of apolymerase upon the nucleotides, according to some embodiments of thepresent invention.

FIGS. 20A-20D schematically illustrate a composition including a tetheranchored to or adjacent to a nanopore and configured for use indetecting action of a polymerase upon a first nucleotide using a changein applied voltage across the nanopore, according to some embodiments ofthe present invention.

FIG. 20E illustrates an exemplary signal that can be generated duringuse of a composition such as illustrated in FIGS. 20A-20D, according tosome embodiments of the present invention.

FIGS. 21A-21D schematically illustrate the composition of FIGS. 20A-20Dconfigured for use in detecting action of the polymerase upon a secondnucleotide using a second change in applied voltage across the nanopore,according to some embodiments of the present invention.

FIG. 21E illustrates an exemplary signal that can be generated duringuse of a composition such as illustrated in FIGS. 21A-21D, according tosome embodiments of the present invention.

FIGS. 22A-22F schematically illustrate a composition including a tetheranchored adjacent to a nanopore and configured for use in detectingaction of a polymerase upon a first nucleotide using a change in appliedvoltage across the nanopore, according to some embodiments of thepresent invention.

FIG. 23A schematically illustrates exemplary nucleotides includingelongated tags including respective reporter regions and moieties thatcan bond to an exemplary tether during use in detecting action of apolymerase upon the nucleotides, according to some embodiments of thepresent invention.

FIG. 23B schematically illustrates a composition including a tetheranchored to or adjacent to a nanopore and configured for use indetecting action of a polymerase upon a first nucleotide based on aninteraction between the tether and a reporter region of a nucleotide,according to some embodiments of the present invention.

FIG. 23C schematically illustrates a detectable interaction between oneof the reporter regions of FIG. 23A with the tether of FIG. 23B duringaction of a polymerase upon a first nucleotide, according to someembodiments of the present invention.

FIGS. 24A-24C illustrate an exemplary protein-DNA tether conjugatecaptured in an MspA nanopore and locked into place using a trans-sidelock oligonucleotide, and FIG. 24D illustrates an exemplary duplexsignal versus time that can be generated using the conjugate illustratedin FIGS. 24A-24C, according to some embodiments of the presentinvention. An oligonucleotide complementary to a region of the DNAtether was then added to the cis side. Voltage was cycled between 120 mVand −60 mV with approximately a 200 msec period. (A) The conjugate uponthe application of forward voltage. Signal is seen (D-2402). (B). Theconjugate upon the application of the negative voltage. Signal is seen(D-2400). (C) Upon hybridization of an oligonucleotide conjugate that ispulled up to the pore constriction. The exemplary signal is seen priorto stripping (D-2401). After stripping, the system returns to the stateshown in FIG. 24A while the voltage is still at 120 mV, resulting insignal D-2402. Data in FIG. 24D was filtered with a 2 KHz low-passfilter for visual clarity.

FIG. 25 illustrates exemplary reaction parameters, e.g., rate constantsand dwell times, for reaction schemes in which a nucleotide respectivelybeing acted upon by a polymerase is a match or a mismatch (adapted fromJohnson, “The kinetic and chemical mechanism of high-fidelity DNApolymerases,” Biochim Biophys Acta 1804(5): 1041-1048 (2010), the entirecontents of which are incorporated by reference herein).

FIGS. 26A-26D illustrate exemplary structures for use in modifying akinetic constant in a reaction scheme in which a nucleotide is beingacted upon by a polymerase, according to some embodiments of the presentinvention.

DETAILED DESCRIPTION

Embodiments of the present invention provide compositions, systems, andmethods for detecting events using tethers anchored to or adjacent tonanopores.

More specifically, the present compositions, systems, and methodssuitably can be used to detect events, such as motion of a molecule or aportion thereof, in a manner that is robust, reproducible, sensitive,and has high throughput. For example, the present compositions caninclude a nanopore and a permanent tether that is anchored to, oradjacent to, the nanopore. The nanopore can include first and secondsides and an aperture that extends through the first and second sides.The permanent tether can include head and tail regions and an elongatedbody disposed therebetween. At least one of the head and tail regions ofthe tether is anchored to, or adjacent to, the first or second side ofthe nanopore. The tether can include one or more features thatfacilitates detection of an event that occurs adjacent to the nanopore,e.g., on the first or second side of the nanopore.

For example, in some embodiments, the elongated body of the tether caninclude a reporter region, e.g., a region that facilitates detection orcharacterization of the tether using a suitable detection technique orapparatus. The reporter region can be movable (e.g., translationally,conformationally, or rotationally movable or a combination thereof)within the aperture responsive to an event that occurs adjacent to thefirst side of the nanopore. The movements of the reporter region aremeasurable, and information about the event is interpretable based onthe measurements of the movements. Additionally, the reporter region canbe configured so as to be repeatedly movable, e.g., responsive todifferent events. Such events can be different from one another, and canoccur in any sequence. In particular embodiments, the events occur in aseries of cycles such as occurs in the synthesis of a polymer bysequential addition of monomers, or in the degradation of a polymer bysequential removal of monomers. Particularly useful polymers are nucleicacids containing nucleotide monomers. Information about each event isindividually determinable based on measurement of the movement of thereporter region responsive to that event. For example, a magnitude or atime duration, or both, of a signal based on the movement of thereporter region can be individually correlated to each event.

One example of an event that can be detected using the presentcompositions, systems, and methods is a conformational change of amolecule that disposed adjacent to the first side of the nanopore.Another example of an event that can be detected using the presentcompositions, systems, and methods is the interaction of one moleculewith another molecule. It should be appreciated that a molecule'sinteraction with another molecule can cause, but need not necessarilycause, a conformational change in one or both of the molecules.Additionally, a conformational change of a molecule can be, but need notnecessarily be, responsive to that molecule's interaction with anothermolecule. The present compositions, systems, and methods can be suitablyconfigured so as to detect any such interaction, or any suchconformational change, or a combination of an interaction andconformational change.

Tethers having other characteristics also can be suitably used to detectevents. For example, the elongated body of the tether can include amoiety that interacts with a first molecule. The event can includeaction upon the first molecule by a second molecule. The tether caninclude a reporter region that is movable (e.g., translationally,conformationally, or rotationally movable or a combination thereof)responsive to the interaction between the moiety of the tether and thefirst molecule. Alternatively, the reporter region of the tether can bedisposed at a location within the aperture that is based upon theinteraction between the moiety of the tether and the first molecule. Asstill another alternative, the first molecule, rather than the tether,can include a reporter region. The presence of such a reporter regioncan be detectable responsive to interaction between the moiety of thetether and the first molecule, or detectable responsive to any othersuitable stimulus. Additionally, the present compositions can be used tostabilize other molecules. For example, the tether's interaction withanother molecule can stabilize that molecule, e.g., temporarily retainthat molecule or a portion thereof within or adjacent to a nanopore.

Other configurations readily can be envisioned based on the teachingsprovided herein.

First, some terms used herein will be briefly explained. Then, someexemplary compositions, exemplary systems including measurementcircuitry (e.g., electrical or optical measurement circuitry) that canbe used with the present compositions, exemplary methods that can beused with the present compositions, and some specific examples ofcompositions that can be used during such methods, will be described.

Exemplary Terms

As used herein, the term “pore” is intended to mean a structure thatincludes an aperture that permits molecules to cross therethrough from afirst side of the pore to a second side of the pore. That is, theaperture extends through the first and second sides of the pore.Molecules that can cross through an aperture of a pore can include, forexample, ions or water-soluble molecules such as nucleic acids,proteins, nucleotides, and amino acids. The pore can be disposed withina barrier. When at least a portion of the aperture of a pore has a widthof 100 nm or less, e.g., 10 nm or less, or 2 nm or less, the pore canbe, but need not necessarily be, referred to as a “nanopore.”Optionally, a portion of the aperture can be narrower than one or bothof the first and second sides of the pore, in which case that portion ofthe aperture can be referred to as a “constriction.” Alternatively oradditionally, the aperture of a pore, or the constriction of a pore (ifpresent), or both, can be greater than 0.1 nm, 0.5 nm, 1 nm, 10 nm ormore. A pore can include multiple constrictions, e.g., at least two, orthree, or four, or five, or more than five constrictions.

As used herein, a “barrier” is intended to mean a structure thatnormally inhibits passage of molecules from one side of the barrier tothe other side of the barrier. The molecules for which passage isinhibited can include, for example, ions or water soluble molecules suchas nucleic acids, proteins, nucleotides, and amino acids. A pore can bedisposed within a barrier, and the aperture of the pore can permitpassage of molecules from one side of the barrier to the other side ofthe barrier. Barriers include membranes of biological origin, andnon-biological barriers such as solid state membranes.

As used herein, “tether” is intended to mean an elongated member havinga head region, a tail region, and an elongated body therebetween. Atether can include a molecule. A tether can be, but need not necessarilybe, in an elongated state, e.g., can include an elongated molecule. Forexample, an elongated body of a tether can have secondary or tertiaryconfigurations such as hairpins, folds, helical configurations, or thelike. Tethers can include polymers such as polynucleotides or syntheticpolymers. Tethers can have lengths (e.g., measured in a stretched ormaximally extended state) ranging, for example, from about 5 nm to about500 nm, e.g., from about 10 nm to about 100 nm. Tethers can have widthsranging, for example, from about 1 nm to about 50 nm, e.g., from about 2nm to about 20 nm. Tethers can be linear or branched. A tether can beconsidered to be “permanent” when it is not removed from a compositionset forth herein under the conditions in which the composition is used,for example, in a detection method. A tether that is used in a cyclic orrepeated reaction can also be considered “permanent” when there is nonet change in position of the tether from one cycle to the next or fromone reaction to a repeat of the reaction. It will be understood that theposition of a permanent tether may change during an individual cycle orreaction even though there is no net change in position across thecycles or reactions.

As used herein, a “head region” of a tether is intended to mean afunctional group of the tether that is attached to another member. Suchattachment can be formed via a chemical bond, e.g., via a covalent bond,hydrogen bond, ionic bond, dipole-dipole bond, London dispersion forces,or any suitable combination thereof. In one embodiment, such attachmentcan be formed through hybridization of a first oligonucleotide of thehead region to a second oligonucleotide of another member.Alternatively, such attachment can be formed using physical orbiological interactions, e.g., an interaction between a first proteinstructure of the head region and a second protein structure of the othermember that inhibits detachment of the head region from the othermember. Exemplary members to which a head region of a tether can beattached include a pore, e.g., the first or second side of the pore, abarrier in which the pore is disposed, and a molecule, such as aprotein, disposed on either the first or second side of the pore. If thehead region of the tether is attached to another member that is disposedon either the first or second side of the pore, the head region of thetether can be said to be adjacent to the pore. The head region can be,but need not necessarily be, located at an end of the tether.

As used herein, “anchored” is intended to mean an attachment between afirst member and a second member that is permanent, e.g., issufficiently stable as to be useful for detecting an event or, e.g., ismovable but undergoes no net movement under the conditions in which theattached members are used. In some embodiments, such a permanentattachment is normally irreversible under the conditions in which theattached members are used, for example, in a detection method. In otherembodiments, such a permanent attachment is reversible but persists forat least the period of time in which it is used for detecting an event.For example, a tether can be permanently attached to or adjacent to apore during use of the tether to detect an event, and can besubsequently removable or replaceable with another tether. Covalentbonds are only one example of an attachment that suitably can be used toanchor a first member to a second member. Other examples includeduplexes between oligonucleotides, peptide-peptide interactions, andstreptavidin-biotin or streptavidin-desthiobiotin.

As used herein, a “tail region” of a tether is intended to mean aportion of the tether that is disposed distally from the head region.The tail region can extend freely away from the head region, e.g., canbe unattached to any other member. The tail region alternatively can beattached. Such attachment can be formed via a chemical bond, e.g., via acovalent bond, hydrogen bond, ionic bond, dipole-dipole bond, Londondispersion forces, or any suitable combination thereof. In oneembodiment, such attachment can be formed through hybridization of afirst oligonucleotide of the tail region to a second nucleotide ofanother member. Alternatively, such attachment can be formed usingphysical or biological interactions e.g., an interaction between a firstprotein structure of the tail region and a second protein structure ofthe other member that inhibits detachment of the tail region from theother member. Any member to which the tail region is attached can be,but need not necessarily be, the same member to which the head region isattached. The tail region can be, but need not necessarily be, locatedat an end of the tether.

As used herein, an “elongated body” is intended to mean a portion of amember, such as a tether, that is sufficiently long and narrow to bedisposed within at least a portion of an aperture of a pore. When anelongated body is attached to a nucleotide being acted upon, such anelongated body can be referred to as an “elongated tag” so as tofacilitate distinction from an elongated body of a tether. An elongatedbody can be formed of any suitable material of biological origin ornonbiological origin, or a combination thereof. In one example, theelongated body includes a polymer. Polymers can be biological orsynthetic polymers. Exemplary biological polymers that suitably can beincluded within an elongated body include polynucleotides, polypeptides,polysaccharides, polynucleotide analogs, and polypeptide analogs.Exemplary polynucleotides and polynucleotide analogs suitable for use inan elongated body include DNA, enantiomeric DNA, RNA, PNA(peptide-nucleic acid), morpholinos, and LNA (locked nucleic acid).Exemplary synthetic polypeptides can include charged amino acids as wellas hydrophilic and neutral residues. Exemplary synthetic polymers thatsuitably can be included within an elongated body include PEG(polyethylene glycol), PPG (polypropylene glycol), PVA (polyvinylalcohol), PE (polyethylene), LDPE (low density polyethylene), HDPE (highdensity polyethylene), polypropylene, PVC (polyvinyl chloride), PS(polystyrene), NYLON (aliphatic polyamides), TEFLON®(tetrafluoroethylene), thermoplastic polyurethanes, polyaldehydes,polyolefins, poly(ethylene oxides), poly(w-alkenoic acid esters),poly(alkyl methacrylates), and other polymeric chemical and biologicallinkers such as described in Hermanson, Bioconjugate Techniques, thirdedition, Academic Press, London (2013). Additionally, an elongated bodyoptionally can include a moiety that can interact with another moiety.Such moieties can include biological polymers DNA, RNA, PNA, LNA,morpholinos, or enantiomeric DNA, for example. Regions of the elongatedbody can be charged or neutral depending on the particularimplementation of the reporter readout.

As used herein, a “reporter region” is intended to mean a moiety thatis, upon relatively small movements, detectable using a suitabledetection method or system. Such movements can be approximately 10 nm orless, or approximately 5 nm or less, or approximately 2 nm or less, orapproximately 1 nm or less, or approximately 0.5 nm or less, orapproximately 0.2 nm or less, or even approximately 0.1 nm or less, andcan be detected using the reporter region and a suitable detectionmethod or system. The moiety can have a detectable physical, chemical,electrical, optical, or biological property or other suitable fluxblockade property. For example, the moiety can have an optical propertythat facilitates optical detection or characterization. Opticalproperties include fluorescence and generation of a Raman signal. In oneillustrative example, the moiety is a fluorescent resonance energytransfer (FRET) donor or acceptor that interacts with a correspondingFRET acceptor or donor so as to emit light of a particular wavelengththat can be detected. The donor and acceptor can be considered to beFRET pair partners. Or, for example, the moiety can have an electricalor flux blockade property. Electrical or flux blockade propertiesinclude electrostatic charge, e.g., a positive charge, or a negativecharge. Or, for example, the moiety can have a physical property.Physical properties include the volume and shape of the moiety. In oneillustrative example, movement of the moiety within the aperture causesa measurable change in current or flux through an aperture, or anoptional constriction therein, by modulating a blockage current or fluxthrough the aperture or constriction. Or, for example, the moiety canhave a chemical or biological property that facilitates chemical orbiological detection. Chemical or biological properties include presenceof a chemical or biological group, e.g., a radioactive group or a grouphaving enzymatic activity. One or more electrical, physical, chemical,biological, or other flux blockade properties of the moiety can providea measurable change in current through an aperture or constriction, ameasurable change in flux of molecules through an aperture orconstriction, or an optical signal. In one illustrative example,movement of the moiety within an aperture causes a measurable change ina current through an aperture or constriction, or causes a measurablechange in flux of molecules through an aperture or constriction, whichchange in flux can be electrically, chemically, biologically, oroptically detectable. An abasic nucleotide is one nonlimiting example ofa moiety the movement of which can cause a measurable change in acurrent through an aperture or constriction or a measurable change influx of molecules through an aperture or constriction.

As used herein, an “event” is intended to mean an action having anassociated effect. In the present context, an action can include, but isnot limited to, the motion of a molecule or a portion of that molecule,and the effect can be any result of such motion. “Motion” or “movement”can be translational, rotational, or conformational, or a combinationthereof. An exemplary effect of such motion can include the movement ofa reporter region within a nanopore aperture or constriction. Forexample, an event can include the translational motion of a molecule, ora rotational change of a molecule, or a conformational change of amolecule. Or, for example, an event can include an interaction between afirst molecule and a second molecule. An exemplary effect associatedwith such an event can be a conformational change of the first molecule,of the second molecule, or of both the first and second molecules. Anevent also can include the concerted action of multiple molecules, orany portion of such concerted action. For example, an event can includea molecule entering an active site on a protein, and the proteinexperiencing a conformational change when acting upon the molecule. Anevent also can include, but is not limited to, a chemical change to amolecule or a portion of that molecule, and the effect can be anyassociated result of such a chemical change. Chemical changes caninclude removing a portion of the molecule, adding the molecule toanother molecule, a first molecule binding or debinding from anothermolecule, modifying the molecule or a portion thereof, and formation orcleavage of a chemical bond, e.g., during polynucleotide synthesis, andthe like. For example, an event can include adding a nucleotide to apolynucleotide, or hybridizing or dehybridizing two oligonucleotides. Anevent optionally can include both motion and chemical change of one ormore molecules. Exemplary effects of any such events can include themovement of a reporter region within a pore aperture or constriction ora reporter region becoming disposed in a particular location within ananopore aperture or constriction. As nonlimiting, purely illustrativeexamples, an event can include one or more of: a polymerase testing anucleotide, the polymerase rejecting a nucleotide if the nucleotide is amismatch to the next nucleotide in a polynucleotide that is beingsequenced, the polymerase excising a nucleotide from a polynucleotideusing exonuclease activity, and the polymerase excising a nucleotidefrom a polynucleotide using pyrophosphorylysis. FIG. 25 illustratesexemplary reaction parameters, e.g., rate constants and dwell times, forreaction schemes in which a nucleotide respectively being acted upon bya polymerase is a match or a mismatch (adapted from Johnson, “Thekinetic and chemical mechanism of high-fidelity DNA polymerases,”Biochim Biophys Acta 1804(5): 1041-1048 (2010), the entire contents ofwhich are incorporated by reference herein). Polymerases such as T7 Poltypically discriminate between match and mismatch nucleotides based on acombination of increased binding affinity for the correct matchnucleotide (e.g., approximately 10-fold preference correct vs.mismatch), greatly reduced catalytic rate for mismatch nucleotide (e.g.,approximately 1000-fold slower for mismatch), and a greatly increasedoff-rate for the mismatch nucleotide from the closed catalytic state(e.g., approximately 300-fold faster for mismatch).

As used herein, a “conformational change” is intended to mean a changein shape of a molecule (e.g., a change in relative atomic coordinates ofa molecule). Such a conformational change can include a portion of amolecule moving relative to another portion of the molecule. Thechemical reactivity of a portion of the molecule can change responsiveto the relative motion of that portion, or another portion, of themolecule. A molecule can undergo a conformational change responsive to astimulus. Such a stimulus can include, but is not limited to, changes toor forces applied to the molecule, interactions with other molecules, orenvironmental factors. Changes to or forces applied to the molecule caninclude a physical force applied to the molecule or a portion thereof,an electrical field applied to the molecule, or a chemical reaction withthe molecule or a portion thereof, or a combination thereof, e.g.,binding of a substrate, catalysis, and/or release of a product.Interactions with other molecules can include the presence of anothermolecule, a concentration of another molecule, an action by or uponanother molecule, or a combination thereof. An exemplary interactionwith another molecule includes hybridization of two oligonucleotides, ora polymerase acting upon a nucleotide. Environmental factors can includea change in pH or a change in temperature, or a combination thereof

As used herein, the term “nucleotide” is intended to mean a moleculethat includes a sugar and at least one phosphate group, and optionallyalso includes a nucleobase. A nucleotide that lacks a nucleobase can bereferred to as “abasic.” Nucleotides include deoxyribonucleotides,modified deoxyribonucleotides, ribonucleotides, modifiedribonucleotides, peptide nucleotides, modified peptide nucleotides,modified phosphate sugar backbone nucleotides, and mixtures thereof.Examples of nucleotides include adenosine monophosphate (AMP), adenosinediphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate(TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP),cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidinetriphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate(GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP),uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosinemonophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosinetriphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidinediphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidinediphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosinemonophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosinetriphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridinediphosphate (dUDP), and deoxyuridine triphosphate (dUTP).

The term “nucleotide” also is intended to encompass any “nucleotideanalogue which is a type of nucleotide that includes a modifiednucleobase, sugar and/or phosphate moiety compared to naturallyoccurring nucleotides. Exemplary modified nucleobases that can beincluded in a polynucleotide, whether having a native backbone oranalogue structure, include, inosine, xathanine, hypoxathanine,isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine,5-hydroxymethyl cytosine, 2-aminoadenine, 6-methyl adenine, 6-methylguanine, 2-propyl guanine, 2-propyl adenine, 2-thiouracil,2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine,5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine,6-azo thymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine,8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituteduracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine,8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine,3-deazaadenine or the like. As is known in the art, certain nucleotideanalogues cannot become incorporated into a polynucleotide, for example,nucleotide analogues such as adenosine 5′-phosphosulfate.

Exemplary nucleotides modified at a phosphate moiety include, forexample, the nucleotide analogues described by Lee et al., “Synthesisand reactivity of novel γ-phosphate modified ATP analogues,” Bioorganic& Medicinal Chemistry Letters 19: 3804-3807 (2009); Kumar et al,“PEG-labeled nucleotides and nanopore detection for single molecule DNAsequencing by synthesis,” Scientific Reports 2: 684 (2012); Kumar etal., “Terminal phosphate labeled nucleotides: synthesis, applications,and linker effect on incorporation by DNA polymerases,” Nucleosides,Nucleotides, and Nucleic Acids 24: 401-408 (2005), and Mulder et al.,“Nucleotide modification at the γ-phosphate leads to the improvedfidelity of HIV-1 reverse transcriptase,” Nucleic Acids Research 33:4865-4873 (2005), the entire contents of which are incorporated byreference herein. Lee et al. describes certain exemplary γ-phosphatemodified ATP analogues having the following structures:

Kumar et al. (2012) discloses different length PEG-coumarin tags whichcan be attached to the terminal phosphate of dNTP or NTP (dNTP/NTP) orto the terminal phosphate of tetraphosphate nucleotides (dN4P/N4P).Exemplary lengths include, for example, coumarin-PEG₁₆-dN4P/N4P,coumarin-PEG₂₀-dN4P/N4P, coumarin-PEG₂₄-dN4P/N4P, andcoumarin-PEG₃₆-dN4P/N4P. Kumar et al. (2005) discloses tetra- andpenta-phosphate-modified nucleotides including dyes attached with orwithout linkers. As described in Kumar et al. (2005) exemplary dyesattached without linkers include DDAO, RESORUFIN, COUMARINS,alkyl-XANTHENES, nitrophenol, hydroxy-indole, ELF, and BBT; exemplarydyes attached via linkers include R110, REG, TAMRA, ROX, Cy dyes, and ETdyes; and exemplary linkers include diaminopropane, diaminoheptane,diaminododecane, EEA, PAP, diaminocyclohexane, diamino-xylene, andpenta-lysine. Mulder et al. discloses chemically modified nucleotidesincluding 1-aminonaphthalene-5-sulfonate (ANS) attached to theγ-phosphate of a nucleotide, e.g., γ-P-aminonaphthalene-5-sulfonatedeoxy or ribonucleotides (dNTP or NTP) such as ANS-ATP, ANS-CTP,ANS-GTP, and ANS-TTP and/or the deoxy forms of these or othernucleotides.

As used herein, the term “polynucleotide” refers to a molecule thatincludes a sequence of nucleotides that are bonded to one another.Examples of polynucleotides include deoxyribonucleic acid (DNA),ribonucleic acid (RNA), and analogues thereof. A polynucleotide can be asingle stranded sequence of nucleotides, such as RNA or single strandedDNA, a double stranded sequence of nucleotides, such as double strandedDNA, or can include a mixture of a single stranded and double strandedsequences of nucleotides. Double stranded DNA (dsDNA) includes genomicDNA, and PCR and amplification products. Single stranded DNA (ssDNA) canbe converted to dsDNA and vice-versa. The precise sequence ofnucleotides in a polynucleotide can be known or unknown. The followingare exemplary examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, expressed sequence tag (EST) or serialanalysis of gene expression (SAGE) tag), genomic DNA, genomic DNAfragment, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomalRNA, ribozyme, cDNA, recombinant polynucleotide, syntheticpolynucleotide, branched polynucleotide, plasmid, vector, isolated DNAof any sequence, isolated RNA of any sequence, nucleic acid probe,primer or amplified copy of any of the foregoing.

As used herein, “hybridize” is intended to mean noncovalently binding afirst polynucleotide to a second polynucleotide. The strength of thebinding between the first and second polynucleotides increases with thecomplementarity between those polynucleotides.

As used herein, the term “protein” is intended to mean a molecule thatincludes, or consists of, a polypeptide that is folded into athree-dimensional structure. The polypeptide includes moieties that,when folded into the three-dimensional structure, impart the proteinwith biological activity.

As used herein, the term “enzyme” is intended to mean a molecule thatcatalytically modifies another molecule. Enzymes can include proteins,as well as certain other types of molecules such as polynucleotides.Examples of enzymes that also are proteins include polymerases,exonucleases and helicases.

As used herein, a “polymerase” is intended to mean an enzyme having anactive site that assembles polynucleotides by polymerizing nucleotidesinto polynucleotides. A polymerase can bind a primed single strandedpolynucleotide template, and can sequentially add nucleotides to thegrowing primer to form a polynucleotide having a sequence that iscomplementary to that of the template.

Exemplary Compositions

Some exemplary compositions including various configurations of tethersanchored to or adjacent to nanopores now will be described withreference to FIGS. 1A-1M. Under one aspect, a composition includes ananopore including a first side, a second side, and an apertureextending through the first and second sides; and a permanent tetherincluding a head region, a tail region, and an elongated body disposedtherebetween. The head region can be anchored to or adjacent to thefirst side or second side of the nanopore.

For example, FIG. 1A schematically illustrates a cross-section of anexemplary composition that includes nanopore 100 and permanent tether110. Nanopore 100 includes first side 101, second side 102, aperture103, and optional constriction 104. Permanent tether 110 includes headregion 111, tail region 112, and elongated body 113. In the embodimentillustrated in FIG. 1A, head region 111 is anchored to first side 101 ofnanopore 100, tail region 112 is disposed on first side 101 of nanopore100 and extends freely toward second side 102 of nanopore 100, andelongated body 113 is movable within aperture 103 of nanopore 100.However, nanopore 100 or tether 110, or both, can have differentconfigurations than illustrated in FIG. 1A, such as exemplified herein.

Head region 111, tail region 112, and elongated body 113 of tether 110can include any suitable material or combination of materials. Forexample, head region 111 can be configured so as to be anchored to firstside 101 via a chemical bond, e.g., via a covalent bond, hydrogen bond,ionic bond, dipole-dipole bond, London dispersion forces, or anysuitable combination thereof. For example, head region 111 can include afirst moiety that is bonded, e.g., covalently, to a second moiety offirst side 101. Exemplary covalent bonds that can anchor head region 111to first side 101 include carbon-carbon bonds, carbon-nitrogen bonds,carbon-oxygen bonds, oxygen-oxygen bonds, sulfur-sulfur bonds,phosphorus-oxygen bonds, phosphorus-sulfur bonds, amide bonds, thioetherbonds, hydrazide bonds, carbon-sulfur bonds, and bonds that result fromthe reaction of oxyamine with carbonyls (aldehydes and ketones), ofStaudinger reagent pairs such as phosphine and azides, or clickchemistry pairs such as azides and alkynes. However, the attachment neednot be covalent. For example, such attachment can be formed throughhybridization of a first oligonucleotide of the head region to a secondnucleotide of another member. Alternatively, such attachment can beformed using physical or biological interactions, e.g., an interactionbetween a first protein structure of the head region and a secondprotein structure of another member that inhibits detachment of the headregion from the other member. For example, head region 111 can include afirst alpha helix and first side 101 can include a second alpha helixthat locks to head region 111 so as to inhibit dissociation of headregion 111 from first side 101. Interactions between receptors andligands are also useful, examples of which include avidin-biotin, oranalogs thereof; antibody-epitope; lectin-carbohydrate, and the like.

Elongated body 113 can be attached, e.g., covalently bonded, to headregion 111, and tail region 112 can define an end of elongated body 113that is distal from head region 111. Elongated body 113 can include anysuitable material of biological origin or a nonbiological origin, or acombination thereof. As described in greater detail below, elongatedbody 113 optionally can include one or more reporter regions thatfacilitate detection or movement of the elongated body, or can includeone or more moieties that interact with other molecules, or can includeone or more of such reporter regions and one or more of such moieties.Other regions of elongated body 113 can be substantially inert, so as toinhibit interaction of such regions with other molecules in a mannerthat otherwise can cause movement of elongated body 113 relative to suchmolecules or relative to nanopore 100. Exemplary biological materialsthat can be included within elongated body 113 include biologicalpolymers such as polynucleotides, polypeptides, polysaccharides, andanalogs of the aforementioned. Exemplary synthetic polymers thatsuitably can be included within elongated body 113 include PEG(polyethylene glycol), PPG (polypropylene glycol), PVA (polyvinylalcohol), PE (polyethylene), LDPE (low density polyethylene), HDPE (highdensity polyethylene), polypropylene, PVC (polyvinyl chloride), PS(polystyrene), NYLON (aliphatic polyamides), TEFLON®(tetrafluoroethylene), thermoplastic polyurethanes, polyaldehydes,polyolefins, poly(ethylene oxides), poly(w-alkenoic acid esters),poly(alkyl methacrylates), and other polymeric chemical and biologicallinkers such as described in Hermanson et al., mentioned further above.

Nanopore 100 can have any suitable configuration that permits anchoringof head region 111 to first side 101 of nanopore 100. In someembodiments, nanopore 100 can be a biological pore, solid state pore, ora biological and solid state hybrid pore. A biological pore is intendedto mean a pore that is made from one or more materials of biologicalorigin. “Biological origin” refers to material derived from or isolatedfrom a biological environment such as an organism or cell, or asynthetically manufactured version of a biologically availablestructure. Biological pores include, for example, polypeptide pores andpolynucleotide pores.

A polypeptide pore is intended to mean a pore that is made from one ormore polypeptides. The one or more polypeptides can include a monomer, ahomopolymer or a heteropolymer. Structures of polypeptide pores include,for example, an α-helix bundle pore and a β-barrel pore as well as allothers well known in the art. Exemplary polypeptide pores includeα-hemolysin, Mycobacterium smegmatis porin A, gramicidin A, maltoporin,OmpF, OmpC, PhoE, Tsx, F-pilus, SP1, mitochondrial porin (VDAC), Tom40,outer membrane phospholipase A, and Neisseria autotransporterlipoprotein (NaIP). “Mycobacterium smegmatis porin A (MspA)” is amembrane porin produced by Mycobacteria, allowing hydrophilic moleculesto enter the bacterium. MspA forms a tightly interconnected octamer andtransmembrane beta-barrel that resembles a goblet and includes a centralconstriction. For further details regarding α-hemolysin, see U.S. Pat.No. 6,015,714, the entire contents of which are incorporated byreference herein. For further details regarding SP1, see Wang et al.,Chem. Commun., 49:1741-1743, 2013, the entire contents of which areincorporated by reference herein. For further details regarding MspA,see Butler et al., “Single-molecule DNA detection with an engineeredMspA protein nanopore,” Proc. Natl. Acad. Sci. 105: 20647-20652 (2008)and Derrington et al., “Nanopore DNA sequencing with MspA,” Proc. Natl.Acad. Sci. USA, 107:16060-16065 (2010), the entire contents of both ofwhich are incorporated by reference herein. Other pores include, forexample, the MspA homolog from Norcadia farcinica, and lysenin. Forfurther details regarding lysenin, see PCT Publication No. WO2013/153359, the entire contents of which are incorporated by referenceherein.

A polynucleotide pore is intended to mean a pore that is made from oneor more nucleic acid polymers. A polynucleotide pore can include, forexample, a polynucleotide origami.

A solid state pore is intended to mean a pore that is made from one ormore materials of non-biological origin. “Solid-state” refers tomaterials that are not of biological origin. A solid-state pore can bemade of inorganic or organic materials. Solid state pores include, forexample, silicon nitride pores, silicon dioxide pores, and graphenepores.

A biological and solid state hybrid pore is intended to mean a hybridpore that is made from materials of both biological and non-biologicalorigins. Materials of biological origin are defined above and include,for example, polypeptides and polynucleotides. A biological and solidstate hybrid pore includes, for example, a polypeptide-solid statehybrid pore and a polynucleotide-solid state pore.

It should be appreciated that different types of nanopores can havedifferent dimensions than one another in multiple respects. For example,as illustrated in FIG. 1A, nanopore 100 can be characterized as having afirst dimension H1 defining a thickness of nanopore 100, e.g., athickness between outer surface 105 of first side 101 and outer surface106 of second side 102, adjacent to aperture 103. In embodiments inwhich nanopore 100 includes optional constriction 104, nanopore 100 alsocan be characterized as having a second dimension H2 defining aconstriction depth, e.g., a depth between outer surface 105 of firstside 101 and the narrowest portion of constriction 104, adjacent toaperture 103. Nanopore 100 also can be characterized as having a firstdiameter D1 defining a diameter of aperture 103, e.g., a diameter ofaperture 103 at the aperture's widest point. In embodiments in whichnanopore 100 includes optional constriction 104, nanopore 100 also canbe characterized as having a second diameter D2 defining a constrictiondiameter, e.g., a diameter of constriction 104 at the constriction'snarrowest point. It should be appreciated that such dimensions ofnanopore 100 should not be construed as limiting, and that otherdimensions of nanopore 100 can be suitably defined. For example, firstdimension H1 of nanopore 100 can vary along the lateral dimension, e.g.,if nanopore 100 includes a relatively thin barrier in which a relativelythick pore is disposed, such as illustrated in FIG. 1K. Or, for example,in embodiments in which nanopore 100 includes optional constriction 104,second dimension H2 of nanopore 100 can vary depending on the relativelocation of constriction 104 to outer surface 105 of first side 101.That is, optional constriction 104 can be located disposed at anysuitable location within nanopore 100, and indeed can even be disposeddistal to first outer surface 105 or outer surface 106 of second side102. FIGS. 1J and 1K, discussed in greater detail below, illustratenon-limiting, exemplary locations of optional constriction 104. Aperture103 and optional constriction 104 need not necessarily be perfectlycircular, and still can be characterized as having an approximatediameter or using any other suitable dimensions. Moreover, nanopore 100can include multiple constrictions, each of which suitably can becharacterized using appropriate dimensions.

In some embodiments, first dimension H1 of nanopore 100 is about 100 nmor smaller, or about 50 nm or smaller, or about 20 nm or smaller, orabout 10 nm or smaller, or about 5 nm or smaller, or about 2 nm orsmaller. For example, H1 can be between about 2 nm and about 100 nm, orbetween about 5 nm and about 50 nm, or between about 10 nm and about 20nm. In embodiments that include optional constriction 104, seconddimension H2 of nanopore 100 is about 100 nm or smaller, or about 50 nmor smaller, or about 20 nm or smaller, or about 10 nm or smaller, orabout 5 nm or smaller, or about 2 nm or smaller, or about 1 nm orsmaller. For example, H2 can be between about 1 nm and about 100 nm, orbetween about 2 nm and about 50 nm, or between about 5 nm and about 20nm. Illustratively, H1 can be between about 5 nm and about 50 nm, and H2(if applicable) can be between about 1 nm and about 5 nm. In oneexemplary embodiment, H1 is about 10 nm and H2 is about 5 nm. In anotherexemplary embodiment, H1 is about 10 nm and H2 is about 6 nm. In anotherexemplary embodiment, H1 is about 10 nm and H2 is about 7 nm. In anotherexemplary embodiment, H1 is about 10 nm and H2 is about 8 nm. In anotherexemplary embodiment, H1 is about 10 nm and H2 is about 9 nm. In anotherexemplary embodiment, H1 is about 10 nm and H2 is about 10 nm. Inanother exemplary embodiment, H1 is about 5 nm and H2 is about 2 nm. Inanother exemplary embodiment, H1 is about 5 nm and H2 is about 3 nm. Inanother exemplary embodiment, H1 is about 5 nm and H2 is about 4 nm. Inanother exemplary embodiment, H1 is about 5 nm and H2 is about 5 nm. Theterms “approximately” and “about” are intended to mean within 10% aboveor below the stated value.

In some embodiments, first diameter D1 of aperture 103 of nanopore 100is about 100 nm or smaller, or about 50 nm or smaller, or about 20 nm orsmaller, or about 10 nm or smaller, or about 5 nm or smaller, or about 2nm or smaller. For example, D1 can be between about 2 nm and about 100nm, or between about 5 nm and about 50 nm, or between about 10 nm andabout 20 nm. In embodiments including optional constriction 104, seconddiameter D2 of constriction 104 of nanopore 100 is about 100 nm orsmaller, or about 50 nm or smaller, or about 20 nm or smaller, or about10 nm or smaller, or about 5 nm or smaller, or about 2 nm or smaller, orabout 1 nm or smaller. For example, D2 can be between about 1 nm andabout 100 nm, or between about 2 nm and about 50 nm, or between about 5nm and about 20 nm. Illustratively, D1 can be between about 5 nm andabout 50 nm, and D2 (if applicable) can be between about 1 nm and about5 nm.

In one illustrative embodiment, D1 is about 5 to 10 nm, and D2 is about1 to 1.2 nm. In another illustrative embodiment, D1 is about 5 to 10 nm,and D2 is about 1.2 to 1.4 nm. In yet another illustrative embodiment,D1 is about 5 to 10 nm, and D2 is about 1.4 to 1.6 nm. In yet anotherillustrative embodiment, D1 is about 5 to 10 nm, and D2 is about 1.6 to1.8 nm. In yet another illustrative embodiment, D1 is about 5 to 10 nm,and D2 is about 1.8 to 2.0 nm. In exemplary embodiments where the poreis MspA, D1 can be, for example, about 4.8 nm, D2 can be, for example,about 1.1 to 1.2 nm, H1 can be, for example, about 9.6 nm, and H2 canbe, for example, about 7.9 to 8.1 nm. In exemplary embodiments where thepore is α-hemolysin, D1 can be, for example, about 2.6 nm, D2 can be,for example, about 1.4 to 1.5 nm, H1 can be, for example, about 10 nm,and H2 can be, for example, about 5 nm. Other suitable combinations ofdimensions suitably can be selected for other types of pores.

The characteristics of permanent tether 110 can be suitably selectedbased on one or more of the dimensions of nanopore 100. For example,elongated body 113 of tether 110 can have a width selected based on D1or D2 (if applicable), or both D1 and D2 (if applicable). For example,the width of elongated body 113 can be selected such that elongated body113 is movable within aperture 103 responsive to an event or otherstimulus, e.g., elongated body 113 has a width that is smaller thanfirst diameter D1 of aperture 103. In embodiments that include optionalconstriction 104, the width of elongated body 113 also can be selectedsuch that at least a portion of elongated body 113 is movable adjacentto constriction 104, e.g., has a width that is equal to, or smallerthan, second diameter D2. Optionally, in embodiments that includeconstriction 104, the width of elongated body 113 also can be selectedsuch that at least a portion of elongated body 113 is movable throughconstriction 104, e.g., has a width that is sufficiently smaller thansecond diameter D2 to permit movement of elongated body 113 throughconstriction 104, e.g., responsive to an event or other stimulus. Ifnanopore 100 includes multiple constrictions (not specificallyillustrated), then the width of elongated body 113 can be selected suchthat elongated body 113 is movable through some or all of suchconstrictions as appropriate.

The length of elongated body 113 of tether 110 can be selected based onH1 or H2 (if applicable), or both H1 and H2 (if applicable). Forexample, the length of elongated body 113 can be selected so as to beshorter than H1, so that tail region 112 would not extend beyond outersurface 106 of the second side 102 of nanopore 100 even if elongatedbody 113 were fully extended through constriction 104 toward second side102. Or, for example, in embodiments including optional constriction104, the length of elongated body 113 can be selected so as to beshorter than H2, so that tail region 112 would not extend beyondconstriction 104 of nanopore 100 even if elongated body 113 were fullyextended toward second side 103. In other embodiments, the length ofelongated body 113 can be selected so as to be longer than H1, so thattail region 112 would extend beyond outer surface 106 of the second side102 of nanopore 100 if elongated body 113 were fully extended throughconstriction 104 toward second side 102. Or, for example, in embodimentsthat include optional constriction 104, the length of elongated body 113can be selected so as to be longer than H2, so that tail region 112would extend beyond constriction 104 of nanopore 100 if elongated body113 were fully extended toward second side 103.

The length of elongated body 113 can be selected so as to permitrelatively free movement of elongated body 113 within aperture 103, atleast on first side 101 of nanopore 100, substantially without sterichindrance or other interference caused by the elongated body itself.That is, elongated body 113 can be configured so as to occupy only aportion of the volume of aperture 103 on first side 101 of nanopore 100,e.g., so as to occupy less than 50% of the volume of aperture 103 onfirst side 101 of nanopore 100, or less than 20% of the volume ofaperture 103 on first side 101 of nanopore 100, or less than 10% of thevolume of aperture 103 on first side 101 of nanopore 100, or less than5% of the volume of aperture 103 on first side 101 of nanopore 100, orless than 1% of the volume of aperture 103 on first side 101 of nanopore100. Additionally, in the embodiment illustrated in FIG. 1A, tail region112 of tether 110 can be unattached to nanopore 100 or to any othermember, thus permitting relatively free movement of the entirety ofelongated body 113 relative to head region 111.

Although FIG. 1A illustrates one exemplary arrangement of the componentsof nanopore 100 and permanent tether 110, it should be understood thatother arrangements suitably can be used. For example, head region 111 ofpermanent tether 110 instead can be anchored to second side 102. Or, forexample, head region 111 of permanent tether 110 instead can be anchoredadjacent to either first side 101 or second side 102 of nanopore 100.Or, for example, tail region 112 of permanent tether 110 instead can bedisposed on second side 102 of nanopore 100. Or, for example, tailregion 112 of permanent tether 110 instead can be anchored to either thefirst side 101 or second side 102 of nanopore 100. Or, for example,elongated body 113 of permanent tether 110 can include a reporterregion, or a moiety that can bond to another molecule, or both areporter region and a moiety that can bond to another molecule. Some ofsuch combinations of features are described herein, but it should beappreciated that all such combinations of features are contemplated andreadily can be envisioned based on the teachings herein.

For example, FIG. 1B illustrates an alternative composition thatincludes nanopore 100 and alternative tether 110′ having head region111′, tail region 112′, and elongated body 113′. Head region 111′ isanchored to first side 101 of nanopore 100. Tail region 112′ extendsfreely toward second side 102 of nanopore 100 in a manner analogous tothat illustrated in FIG. 1A, except that elongated body 113′ issufficiently long that tail region 112′ can be disposed on second side102 of nanopore 100. Constriction 104 is optional.

Under another aspect, a composition includes a nanopore including afirst side, a second side, and an aperture extending through the firstand second sides; and a permanent tether including a head region, a tailregion, and an elongated body disposed therebetween. The head region canbe anchored to or adjacent to the first side or second side of thenanopore. The elongated body including a reporter region can be movablewithin the aperture responsive to a first event occurring adjacent tothe first side of the nanopore. The reporter region can betranslationally movable within the aperture responsive to the firstevent. Additionally, or alternatively, the reporter region can berotationally movable within the aperture responsive to the first event.Additionally, or alternatively, the reporter region can beconformationally movable within the aperture responsive to the firstevent.

For example, FIG. 1C illustrates an alternative composition thatincludes nanopore 100 and alternative tether 110″ having head region111″, tail region 112″, and elongated body 113″. Head region 111″ isanchored to first side 101 of nanopore 100. Tail region 112″ extendsfreely toward second side 102 of nanopore 100, and elongated body 113″is sufficiently long that tail region 112″ can be disposed on secondside 102 of nanopore 100. Additionally, elongated body 113″ includes afirst reporter region 114″, which facilitates measurement of thetranslational, rotational, or conformational movement of elongated body113″, e.g., movement relative to optional constriction 104 inembodiments that include such a constriction. For example, firstreporter region 114″ can have a different physical, chemical, optical,electrical, biological, or other suitable flux blockade property thanone or more other regions of elongated body 113″. Translational,rotational, or conformational movement of first reporter region 114″,represented in FIG. 1C by the dashed arrow, can be detectable using oneor more techniques described herein, known in the art, or yet to bedeveloped. Optionally, elongated body 113″ can include more than onereporter region, e.g., can include second reporter region 114′″.Elongated body 113″ can include any suitable number of reporter regions,e.g., one, or two, or three, or four, or five, or more than fivereporter regions. Each such reporter region can be the same as eachother reporter region. Alternatively, each such reporter region can bedifferent than each other reporter region. Or, some reporter regions canbe the same as one another, while other reporter regions can bedifferent than one another.

In certain embodiments, first reporter region 114″ and optional secondreporter region 114′″ are translationally movable toward first side 101of nanopore 100 responsive to a first event. First reporter region 114″and optional second reporter region 114′″ also can be translationallymovable toward second side 102 of nanopore 100 after the first event.First reporter region 114″ and optional second reporter region 114′″also can be translationally movable toward first side 101 of nanopore100 responsive to a second event after the first event, and againtranslationally movable toward second side 102 of nanopore 100 after thesecond event. The first or second event, or both, can occur adjacent tothe first side of the nanopore. In embodiments that include optionalconstriction 104, first reporter region 114″ can be disposed at alocation along elongated body 113″ that is selected such that, basedupon elongated body 113″ being fully or partially extended, firstreporter region 114″ is positionable adjacent to or within constriction104. Additionally, optional second reporter region 114′″ can be disposedat a location along elongated body 113″ that is selected such that,based upon elongated body 113″ being fully or partially extended, secondreporter region 114″ is positionable adjacent to or within constriction104. In some embodiments, first reporter region 114′″ is positionableadjacent to or within constriction 104 responsive to a first event, andsecond reporter region 114′″ is positionable adjacent to or withinconstriction 104 responsive to a second event, and the first and secondevents are distinguishable from one another based on detecting whetherthe first reporter region 114″ or the second reporter region 114′″ isdisposed adjacent to or within constriction 104. In one illustrative,nonlimiting example, elongated body 113″ includes a polynucleotide thatincludes one or more abasic nucleotides that define first reporterregion 114″ and optional second reporter region 114′″ along a portion ofthe length of elongated body 113″. An abasic nucleotide can be detectedwithin an aperture of a nanopore as described, for example, in Wilson,“Electronic Control of DNA Polymerase Binding and Unbinding to SingleDNA Molecules Tethered in a Nanopore,” Ph.D. Thesis, University ofCalifornia Santa Cruz (2009), the entire contents of which areincorporated by reference herein. Illustratively, movement or presenceof one or more abasic nucleotides or other suitable reporter region(s)114″, 114′″ can cause a measurable change in a current through aperture103 or constriction 104, a measurable change in flux of moleculesthrough aperture 103 or constriction 104, or an optical signal. Forexample, a change in a flux of molecules through aperture 103 orconstruction 104 can be detected electrically, chemically, biologically,or optically.

As another example, FIG. 1D illustrates an alternative composition thatincludes nanopore 100 and alternative tether 120 having head region 121,tail region 122, and elongated body 123. Head region 121 is anchored tosecond side 102 of nanopore 100. Tail region 122 extends freely towardfirst side 101 of nanopore 100, and elongated body 123 is sufficientlylong that tail region 122 can be disposed on first side 101 of nanopore100. However, it should be appreciated that tail region 122 instead canbe disposed on second side 102 of nanopore 100, e.g., that elongatedbody 123 is of such a length that tail region 122 is disposed on secondside 102 of nanopore 100 even if elongated body 123 is fully extended.Additionally, elongated body 123 includes reporter region 124, whichfacilitates measurements of translational, rotational, or conformationalmovement (or a combination thereof) of elongated body 124, e.g., asrepresented in FIG. 1D by the dashed arrow. In certain embodiments,reporter region 124 is translationally movable toward first side 101 ofnanopore 100 responsive to a first event or other stimulus, andtranslationally movable toward second side 102 of nanopore 100 after thefirst event or other stimulus. Reporter region 124 also can betranslationally movable toward first side 101 of nanopore 100 responsiveto a second event or other stimulus after the first event or otherstimulus, and again movable toward second side 102 of nanopore 100 afterthe second event or other stimulus. The first or second event, or both,can occur adjacent to the first side of the nanopore. The stimulus caninclude, for example, an applied voltage across nanopore 100. Inembodiments that include optional constriction 104, reporter region 124can in some embodiments be movable adjacent to or even throughconstriction 104, e.g., responsive to an event or other stimulus. Itshould be appreciated that elongated body 123 need not necessarilyinclude reporter region 124.

As another example, FIG. 1E illustrates an alternative composition thatincludes nanopore 100 and alternative tether 120′ having head region121′, tail region 122′, and elongated body 123′. Head region 121′ isanchored adjacent to first side 101 of nanopore 100, e.g., is anchoredto another member 150′ that can have, but need not necessarily have, asubstantially fixed position relative to nanopore 100, and can bedisposed adjacent to nanopore 100. Tail region 122′ extends freelytoward second side 102 of nanopore 100 in a manner analogous to thatillustrated in FIG. 1A. In the embodiment illustrated in FIG. 1E, tailregion 122′ is disposed on first side 101 of nanopore 100, e.g.,elongated body 123′ has a length selected such that tail region 122′ isdisposed on first side 101 of nanopore 100 even if elongated body 123′is fully extended. It should be appreciated that elongated body 123′instead can be sufficiently long that tail region 122′ can be disposedon second side 102 of nanopore 100. Constriction 104 is optional. Headregion 121′ instead can be anchored to another member (not illustrated)disposed adjacent to second side 102 of nanopore 100.

As another example, FIG. 1F illustrates an alternative composition thatincludes nanopore 100 and alternative tether 120″ having head region121″, tail region 122″, and elongated body 123″. Head region 121″ isanchored adjacent to first side 101 of nanopore 100, e.g., is anchoredto another member 150″ that can have, but need not necessarily have, asubstantially fixed position relative to nanopore 100, and can bedisposed adjacent to nanopore 100. Tail region 122″ extends freelytoward second side 102 of nanopore 100, and elongated body 123″ issufficiently long that tail region 122″ can be disposed on second side102 of nanopore 100. Additionally, elongated body 123″ includes reporterregion 124″, which facilitates measurement of translational, rotational,or conformational movement of elongated body 113″, e.g., as representedin FIG. 1F by the dashed arrow. In certain embodiments, reporter region124″ is translationally movable toward first side 101 of nanopore 100responsive to a first event, and translationally movable toward secondside 102 of nanopore 100 after the first event. Reporter region 124″also can be translationally movable toward first side 101 of nanopore100 responsive to a second event after the first event, and againtranslationally movable toward second side 102 of nanopore 100 after thesecond event. The first or second event, or both, can occur adjacent tothe first side of the nanopore. In embodiments that include constriction104, reporter region 124″ can be translationally movable adjacent to oreven through constriction 104, e.g., responsive to an event or otherstimulus. Head region 121″ instead can be anchored to another member(not illustrated) disposed adjacent to second side 102 of nanopore 100.

The lengths of the present elongated bodies suitably can be varied suchthat the present tail regions can be disposed at any suitable locationrelative to nanopore 100. For example, FIG. 1G illustrates analternative composition that includes nanopore 100 and alternativetether 130 having head region 131, tail region 132, and elongated body133. Head region 131 is anchored to first side 101 of nanopore 100. Tailregion 132 extends freely toward second side 102 of nanopore 100, andelongated body 133 is sufficiently long that tail region 132 can bedisposed beyond second side 102 of nanopore 100, e.g., beyond outersurface 106 of second side 102. Optionally, elongated body 133 alsoincludes reporter region 134. Head region 131 instead can be anchored tosecond side 102 of nanopore 100, or adjacent to either the first side101 or second side 102 of nanopore 100.

Additionally, the present tail regions need not necessarily extendfreely, but instead can be attached to any suitable member. For example,FIG. 1H illustrates an alternative composition that includes nanopore100 and alternative tether 130′ having head region 131′, tail region132′, and elongated body 133′. Head region 131′ is anchored to firstside 101 of nanopore 100, although head region 131′ instead can beanchored adjacent to first side 101 of nanopore 100. Tail region 132′extends through aperture 103 of nanopore 100, and is anchored on secondside 102 of nanopore 100, e.g., is anchored to outer surface 106 ofsecond side 102, although tail region 132′ instead can be anchoredadjacent to second side 102 of nanopore 100. Elongated body 133′ issufficiently long to permit attachment of head region 131′ to oradjacent to first side 101 of nanopore 100 and attachment of tail region132′ to or adjacent to second side 102 of nanopore 100. Optionally,elongated body 133 also includes reporter region 134′. Alternatively,head region 131′ can be attached to, or adjacent to, second side 102 ofnanopore 100 and tail region 132′ can be attached to, or adjacent to,first side 101 of nanopore 100.

As another example, FIG. 1I illustrates an alternative composition thatincludes nanopore 100 and alternative tether 130″ having head region131″, tail region 132″, and elongated body 133″. Head region 131″ isanchored to first side 101 of nanopore 100, although head region 131″instead can be anchored adjacent to first side 101 of nanopore 100. Tailregion 132″ extends through aperture 103 of nanopore 100, and isattached adjacent to or beyond second side 102 of nanopore 100, e.g., isanchored to another member 150′″ that is disposed adjacent to, orbeyond, outer surface 106 of second side 102. Alternatively, member150′″ can be fully or partially disposed within aperture 103. Elongatedbody 133″ is sufficiently long to permit attachment of head region 131″to or adjacent to first side 101 of nanopore 100 and attachment of tailregion 132″ to member 150′″, e.g., adjacent to or beyond second side 102of nanopore 100, or within aperture 103. Optionally, elongated body 133also includes reporter region 134″. Alternatively, head region 131″ canbe attached to or adjacent to second side 102 of nanopore 100 and tailregion 132″ can be attached adjacent to or beyond first side 101 ofnanopore 100, e.g., can be anchored to another member (not illustrated)that is disposed adjacent to, or beyond, outer surface 105 of first side101.

It should be appreciated that any suitable type of nanopore and anysuitable type of permanent tether can be used in the embodimentsillustrated in FIGS. 1A-1I. For example, as noted further above, thenanopore can include a biological pore, solid state pore, or abiological and solid state hybrid pore. FIG. 1J illustrates an exemplarycomposition that includes solid state nanopore 100′ and tether 140having head region 141, tail region 142, and elongated body 143.Nanopore 100′ includes first side 101′ and second side 102′ that caninclude any suitable solid state material or combination of solid statematerials. First side 101′ can be defined by layer 107′ that includesone or more solid state materials, and is disposed upon second side102′, which can include one or more solid state materials. Exemplarysolid state materials suitable for use in first side 101′ or second side102′, or both, include silicon (Si), silicon nitride (SiN or SiN_(x)),graphene, and silicon oxide (SiO₂ or SiO_(x)). In the illustratedembodiment, aperture 103′ can be defined through second side 102′, andconstriction 104′ can be defined through layer 107′. However, it shouldbe appreciated that layer 107′ and second side 102′ can have anysuitable configurations so as to define an aperture and a constrictionregion. Head region 141 is anchored to outer surface 105′ of first side101′ of nanopore 100′, although head region 141 instead can be anchoredadjacent to first side 101′ of nanopore 100′, or can be anchored to oradjacent to second side 102′ of nanopore 100′. In the embodimentillustrated in FIG. 1J, tail region 142 extends freely toward secondside 102′ of nanopore 100′, and elongated body 143 is sufficiently longthat tail region 142 can be disposed on second side 102′ of nanopore100′. Alternatively, tail region 142 can extend freely toward, or can beattached to, adjacent to, or beyond, either of the first side 101′ orsecond side 102′ of nanopore 100′. Optionally, elongated body 143 alsoincludes reporter region 144. For further details regarding solid statenanopores, see the following references, the entire contents of each ofwhich are incorporated by reference herein: Dekker, “Solid-statenanopores,” Nature Nanotechnology 2: 209-215 (2007); Schneider et al.,“DNA Translocation through Graphene Nanopores,” Nano Letters 10:3163-3167 (2010); Merchant et al. Nano Letters 10:2915-2921 (2010); andGaraj et al., “Graphene as a subnanometre trans-electrode membrane,”Nature 467: 190-193 (2010).

As another example, FIG. 1K illustrates an exemplary composition thatincludes biological or biological and solid state hybrid nanopore 100″and tether 140′ having head region 141′, tail region 142′, and elongatedbody 143′. Nanopore 100″ includes barrier 107″ and biological pore 108″disposed within barrier 107″. Biological pore 108″ includes aperture103″ defined therethrough, and one or more constrictions 104″.Biological pores include, for example, polypeptide pores andpolynucleotide pores. Barrier 107″ can include a membrane of biologicalorigin, or a solid state membrane. Membranes of biological origininclude lipid bilayers. Solid state membranes include silicon andgraphene. Head region 141′ of tether 140′ is anchored to or adjacent tofirst side 101″ of nanopore 100″. For example, in the embodimentillustrated in FIG. 1K, head region 141′ is anchored to biological pore108″ on the first side 101″ of nanopore 100″, e.g., covalently bonded toa moiety on biological pore 108″ on first side 101″. Head region 141′instead can be anchored adjacent to first side 101″ of nanopore 100″,e.g., can be anchored to a member that is adjacent to biological pore108″ on first side 101″, or can be anchored to or adjacent to secondside 102″ of nanopore 100″. Tail region 142′ extends freely towardsecond side 102″ of nanopore 100″, and elongated body 143′ issufficiently long that tail region 142′ can be disposed on or beyondsecond side 102″ of nanopore 100″, e.g., beyond outer surface 106″ ofbarrier 107″. Alternatively, tail region 142′ can extend freely toward,or can be attached to, adjacent to, or beyond, either of the first side101″ or second side 102″ of nanopore 100″. Optionally, elongated body143′ also includes reporter region 144′. For further details regardingexemplary hybrid nanopores and the preparation thereof, see thefollowing references, the entire contents of each of which areincorporated by reference herein: Hall et al., “Hybrid pore formation bydirected insertion of alpha hemolysin into solid-state nanopores,”Nature Nanotechnology 5: 874-877 (2010), and Cabello-Aguilar et al.,“Slow translocation of polynucleotides and their discrimination byα-hemolysin inside a single track-etched nanopore designed by atomiclayer deposition,” Nanoscale 5: 9582-9586 (2013).

Note that in any of the embodiments described herein, the nanopore neednot necessarily include the optional constriction. For example, FIG. 1Lillustrates an exemplary composition that includes alternative nanopore100′″ and tether 140″ having head region 141″, tail region 142″, andelongated body 143″. Nanopore 100′″ includes first side 101′″ and secondside 102′″ that can include any suitable solid state material orcombination of solid state materials. First side 101′″ and second side102′″ can be defined by layer 107′″ that includes one or more solidstate materials. Exemplary solid state materials suitable for use inlayer 107′″ include silicon (Si), silicon nitride (SiN or SiN_(x)),graphene, silicon oxide (SiO₂ or SiO_(x)), or a combination thereof. Inthe illustrated embodiment, aperture 103′″ can be defined through firstand second sides 101′″, 102′″, and can lack a constriction region. Headregion 141″ of tether 140″ is anchored to outer surface 105′″ of firstside 101′″ of nanopore 100′″, although head region 141″ instead can beanchored adjacent to first side 101′″ of nanopore 100′″, or can beanchored to or adjacent to second side 102′″ of nanopore 100′. In theembodiment illustrated in FIG. 1L, tail region 142″ extends freelytoward second side 102′″ of nanopore 100′″, and elongated body 143″ issufficiently long that tail region 142″ can be disposed on second side102′″ of nanopore 100′″. Alternatively, tail region 142″ can extendfreely toward, or can be attached to, adjacent to, or beyond, either ofthe first side 101′″ or second side 102′″ of nanopore 100′″.

Optionally, elongated body 143″ also includes reporter region 144″.Reporter region 144″ can facilitate measurement of translational,rotational, or conformational movement of elongated body 143″. In oneexemplary embodiment, dimension D1 of aperture 103″ suitably is selectedso as to facilitate the use of reporter region to measure movement ofelongated body 143″. For example, aperture 103″ can be sufficientlynarrow so as to measurably interact with reporter region 144″ responsiveto movement of reporter region 144″. As one example, reporter region144″ has an electrical or flux blockade characteristic, and aperture103″ is has a width selected such that movement of reporter region 144″causes a detectable change in current or flux through aperture 103″under an applied voltage across nanopore 100′″. For example, nucleotidesthat are larger (such as A and G) can result in more blockage when theyare disposed in an aperture, e.g., disposed in the constriction of MspA,as compared with T, which is smaller. Exemplary ranges of blockagecurrents or fluxes in terms of % of open pore current or flux include 0to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 60% to 70%, 70%to 80%, 80% to 90%, and 90% to 100%. In one exemplary embodiment, therange is between 20% and 70% for MspA in 300 mM KCL with a 180 mV biasand an open pore current of 110 pA.

In yet another example, FIG. 1M illustrates an alternative compositionthat includes nanopore 100 and alternative tether 170 having head region171, tail region 172, and elongated body 173. Head region 171 isanchored adjacent to first side 101 of nanopore 100, e.g., is anchoredto another member 180 that optionally may have, but need not necessarilyhave, a substantially fixed position relative to nanopore 100. Tailregion 172 extends through aperture 103 of nanopore 100, and is attachedadjacent to or beyond second side 102 of nanopore 100, e.g., is anchoredto another member 180′ that is disposed within aperture 103 or isdisposed adjacent to, or beyond, the outer surface of second side 102.Elongated body 173 is sufficiently long to permit attachment of headregion 171 to member 180 and attachment of tail region 172 to member180′. Optionally, member 180 is sufficiently large as to be unable tophysically pass through the entirety of aperture 103. Additionally, oralternatively, member 180′ is sufficiently large as to be unable tophysically pass through the entirety of aperture 203. Accordingly, theattachment of head region 171 to member 180 and attachment of tailregion 172 to member 180′ can retain tether 170 in nanopore 100, canretain member 180 on first side 101 of nanopore 100, and can retainmember 180′ on second side 102 of nanopore 100, even if members 180 and180′ are not respectively attached to first side 101 or second side 102of nanopore 100. Accordingly, head region 171 can be considered to beanchored adjacent to first side 101 regardless of whether member 180 isattached to first side 101. In one example, the composition illustratedin FIG. 1M can be prepared by attaching head region 171 to member 180,followed by disposing tail region 172 on second side 102, followed byattaching tail region 172 to member 180′. In another example, thecomposition illustrated in FIG. 1M can be prepared by attaching tailregion 172 to member 180′, followed by disposing head region 171 onfirst side 101, followed by attaching head region 171 to member 180. Anysuitable attachment, including those described elsewhere herein, can beused. In one non-limiting, purely illustrative embodiment, first member180 can include a polymerase, and second member 180′ can include anucleic acid that hybridizes to a nucleic acid of tail region 172. Inone example, an exemplary preparation of such a composition, and anexemplary use of such a composition to detect action of the polymeraseupon a nucleotide, are described in greater detail herein with referenceto FIGS. 22A-22D.

Additionally, elongated body 173 optionally includes reporter region174, which facilitates measurement of translational, rotational, orconformational movement of elongated body 173, e.g., as represented inFIG. 1M by the dashed arrow. In certain embodiments, reporter region 174is translationally movable toward first side 101 of nanopore 100responsive to a first event, and translationally movable toward secondside 102 of nanopore 100 after the first event. Reporter region 174 alsocan be translationally movable toward first side 101 of nanopore 100responsive to a second event after the first event, and againtranslationally movable toward second side 102 of nanopore 100 after thesecond event. The first or second event, or both, can occur adjacent tothe first side of the nanopore. In embodiments that include constriction104, reporter region 174 can be translationally movable adjacent to oreven through constriction 104, e.g., responsive to an event or otherstimulus.

Additionally, note that in any of the foregoing examples, as well asother compositions not specifically illustrated, the elongated body ofthe tether optionally can include a moiety that interacts with amolecule. Such interaction can, for example, cause a change in therelative position of a reporter region so as to measurably indicate thepresence of the molecule, or can stabilize the molecule in a particularposition relative to the constriction of the nanopore. Some nonlimitingexamples of such moieties, and uses thereof, are provided furtherherein.

Additionally, it should be appreciated that a head group of a tether canbe attached to a nanopore in any number of ways. For example, well-knownbioconjugate chemistry such as described by Hermanson, mentioned above,can be used. In illustrative embodiments, the nanopore includes achemical moiety for forming an attachment such as a cysteine, or apeptide linker such as a SpyTag. Further information regarding spytagsand use thereof to form attachments can be found, for example, in thefollowing references, the entire contents of each of which areincorporated by reference herein: Zakeri et al., “Peptide tag forming arapid covalent bond to a protein, through engineering a bacterialadhesin,” Proc. Nat. Acad. Sci. USA 109: E690-E697 (2012), and Fierer etal., “SpyLigase peptide-peptide ligation polymerases affibodies toenhance magnetic cancer cell capture,” Proc. Nat. Acad. Sci. USA 111:E1176-E1181 (2014).

Moreover, it should be appreciated that another member (to which thehead group of the tether can be attached) can be attached to or adjacentto a nanopore in any number of ways. For example, the head group of atether can be attached to another member, then the other member can beloaded onto or adjacent to the nanopore, and the other member then canbe attached to or adjacent to the nanopore using a suitable attachment.In one nonlimiting, purely illustrative example, the head group of atether can be attached to a polymerase, then the polymerase can beloaded onto or adjacent to the nanopore, and the polymerase then can beattached to or adjacent to the nanopore using a suitable attachment,such as a covalent bioconjugated linker between the tether and thenanopore. In this manner, the tether can be attached to the polymerase,and both the tether and the polymerase can be attached to the nanoporevia a linkage on the tether. Examples of such linkers include:NHS-esters, isocyanates, and isothicyanate linker conjugation to amines,maleimides to cysteines, Click-chemistry with azides to alkynes, use offusion tags such as Halotag, Spycatcher-Spytag, and other similarprotein-protein bioconjugation methods. For further information aboutexemplary linkages that can be used, see the following references, theentire contents of each of which are incorporated by reference herein:Hermanson, Bioconjugate Techniques, 2nd Ed., Elsevier, 2008; Zakeri etal., “Peptide tag forming a rapid covalent bond to a protein, throughengineering a bacterial adhesin,” PNAS 109(12): E691-E697 (2012); andLiu et al., “Specific Enzyme Immobilization Approaches and TheirApplication with Nanomaterials,” Topics in Catalysis 55(16-18):1146-1156 (2012).

Exemplary Systems

Exemplary systems for detecting events using tethers anchored to oradjacent to nanopores now will be described with reference to FIGS.2A-2C. FIG. 2A schematically illustrates a system including measurementcircuitry (e.g., electrical or optical measurement circuitry) configuredto measure movement or presence of a reporter region within the apertureof a nanopore. System 220 includes nanopore 200, permanent tether 210,and measurement circuit 230. Nanopore 200 includes first side 201,second side 202, aperture 203, and optionally also includes constriction204. Permanent tether 210 includes head region 211, tail region 212, andelongated body 213. In the embodiment illustrated in FIG. 2A, headregion 211 is anchored to first side 201 of nanopore 200, tail region212 is disposed on second side 201 of nanopore 200 and extends freelytoward second side 202 of nanopore 100 or is attached to another member,and elongated body 213 is movable through aperture 203 of nanopore 200.However, nanopore 200 or tether 210, or both, can have differentconfigurations than illustrated in FIG. 2A, such as exemplified herein.For example, head region 211 can be anchored to or adjacent to nanopore200, for example, using a thioether or amide linkage. In oneillustrative, nonlimiting example, a thioether linkage can be created bya maleimide group on tether 211 that reacts with a reduced thiol groupin a cysteine residue on or adjacent to nanopore 200. Introduction of amaleimide group into tether 211 can be readily achieved using methodswell known in the art. Illustratively, head region 211 can be attachedto another member (e.g., a polymerase) disposed on or adjacent to firstside 201 of nanopore 200 in a manner analogous to that described abovewith reference to FIGS. 1F and 1M, or tail region 212 can be attached toanother member (e.g., a nucleic acid) disposed on second side 202 ofnanopore 200 (e.g., within aperture 203) in a manner analogous to thatdescribed above with reference to FIGS. 1I and 1M, or both head region211 can be attached to another member (e.g., a polymerase) disposed onor adjacent to first side 201 of nanopore 200 and tail region 212 can beattached to another member (e.g., a nucleic acid) disposed on secondside 202 of nanopore 200 (e.g., within aperture 203) in a manneranalogous to that described above with reference to FIG. 1M. Optionally,one or both of such members can be sufficiently large as to be unable topass entirely through aperture 203 of nanopore 200.

Additionally, elongated body 213 can include reporter region 214 thatfacilitates measurement of translational, rotational, or conformationalmovement or presence (or a combination thereof) of elongated body 213using measurement circuit 230. For example, reporter region 214 can havea different physical, chemical, electrical, optical, biological, orother suitable flux blockade property than one or more other regions ofelongated body 213. In some embodiments, measurement circuit 230 can beconfigured to optically, electrically, chemically, or biologicallydetect movement of reporter region 214 relative to constriction 204,e.g., as represented in FIG. 2A by the dashed arrow. For example, asystem can include a composition and measurement circuitry configured tomeasure current or flux through the aperture or an optical signal whilethe reporter region of a tether is moved responsive to an event. In oneillustrative example, nanopore 200 and tether 210 can be immersed in aconductive fluid, e.g., an aqueous salt solution. Measurement circuit230 can be in communication with first electrode 231 and secondelectrode 232, and can be configured to apply a voltage between firstelectrode 231 and second electrode 232 so as to impose a voltage acrossnanopore 200. Either a direct-current (DC) or an alternating-current(AC) voltage suitably can be used. In some embodiments, measurementcircuit 230 further can be configured to use first electrode 231 andsecond electrode 232 to measure the magnitude of a current or fluxthrough aperture 203. In some embodiments, measurement circuit 230further can include an optical, biological, or chemical sensorrespectively configured to optically, biologically, or chemically sensethe magnitude of a molecular flux through aperture 203. Exemplaryoptical sensors include CCDs and photodiodes. In some embodiments,measurement circuit 230 includes one or more agents that chemically orbiologically react with the molecular flux through aperture 203 so as togenerate an optically detectable signal.

For example, reporter region 214 can have a different physical propertythan some or all other regions of elongated body 213. For example,reporter region 214 can cause a differential blockage current or fluxthrough aperture 203 as compared to other regions of elongated body 213.Additionally, or alternatively, reporter region 214 can have a differentelectrical or flux blockade property than some or all other regions ofelongated body 213. For example, reporter region 214 can include anelectrostatic charge, while some or all other regions of elongated body213 can include a different electrostatic charge, or can be uncharged(e.g., can be electrically neutral). Or, for example, reporter region214 can be uncharged, while some or all other regions of elongated body213 can include an electrostatic charge. Or, for example, reporterregion 214 can have a physical property. Physical properties include thevolume and shape of reporter region 214. In one illustrative example,movement of reporter region 214 within aperture 203 causes a measurablechange in current or flux through the aperture, or optional constriction204 therein, by modulating a blockage current or flux through theaperture or constriction. Or, for example, reporter region 214 can havea chemical or biological property that facilitates chemical orbiological detection. Chemical or biological properties include presenceof a chemical or biological group, e.g., a radioactive group or a grouphaving enzymatic activity.

One or more electrical, physical, chemical, optical, biological, orother flux blockade properties of reporter region 214 can provide ameasurable change in current through aperture 203 or constriction 204, ameasurable change in flux of molecules through aperture 203 orconstriction 204, or an optical signal. In one illustrative example,movement or presence of reporter region 214 within aperture 203 causes ameasurable change in a current through aperture 203 or constriction 204,or causes a measurable change in flux of molecules through aperture 203or constriction 204, which change in flux can be electrically,chemically, biologically, or optically detectable. For example, presenceor movement of reporter region 214 within aperture 203 or constriction204 can cause an ionic current blockade or a molecular flux blockade,which can be detected optically, electrically, chemically, orbiologically. Illustratively, a gradient of a molecule on the trans sidecan create a natural molecular flux that can be partially blocked byreporter region 214. Measurement circuitry 230 can be configured tomeasure such a molecular flux non-electrically (e.g., optically) usingfluxes of luminescent (e.g., fluorescent or chemiluminescent molecules,or fluxes of reagents that become chemiluminescent in the presence ofother reagents. For example, Ca²⁺ can flux from one side of the nanoporeto the other side where it encounters a calcium sensitive dye, such asFluo-2, Fluo-4, Fluo-8, or the like, to induce fluorescence. Otherreagent pairs that can be used include, but are not limited to, luminoland oxidants, calcium and aequorin, or ATP and luciferase, to name afew. For further details regarding optical detection of molecular fluxesthrough an aperture or constriction, see Ivankin et al., “Label-FreeOptical Detection of Biomolecular Translocation through NanoporeArrays,” ACSNano 8(10): 10774-10781 (2014), the entire contents of whichare incorporated by reference herein.

Illustratively, the magnitude of the current or flux through aperture203 or optical signal can measurably change responsive to movement ofreporter region 214 within aperture 203, and the time period for such ameasurable change in the current or flux or optical signal is based onthe duration of the reporter region's change in position. In oneillustrative, non-limiting example, elongated body 213 includes apolynucleotide that includes one or more abasic nucleotides that definereporter region 214.

In one illustrative embodiment, nanopore 200 is a biological nanopore towhich tether 211 is attached using a thioether linkage. Non-limitingexamples of biological nanopores include MspA and alpha hemolysin.Reporter region 214 of tether 211 can include one or more abasicresidues configured to be positioned within or adjacent to one or moreconstrictions 204 of the biological nanopore. Movement of one or moreproperly positioned abasic residues through a constriction of eitherpore can result in a readily detectable signal, e.g., a detectablechange in current or flux through the constriction(s) 204 or an opticalsignal. Biological nanopores such as MspA and alpha hemolysin usefullycan include constrictions that can serve to focus the effect of reporterregion 214. For example, MspA includes a single constriction with adiameter of approximately 1.2 nm and a length of approximately 0.5 nm,can provide suitable spatial resolution because the magnitude of theionic current or flux blockade through the constriction primarily arebased on the elongated-body segment threaded through the narrow region(constriction) of the nanopore.

FIG. 2B is a plot of an exemplary signal (e.g., optical or electricalsignal) that system 220 illustrated in FIG. 2A can generate as reporterregion 214 translationally, rotationally, or conformationally moves overtime, e.g., moves responsive to one or more events or other stimulus.The value (e.g., magnitude) of the signal at time t₀ can correspond to afirst translational, rotational, or conformational position of reporterregion 214 within aperture 203. At time t₁, the value (e.g., magnitude)of signal can change to a second value, corresponding to reporter region214 translationally, rotationally, or conformationally moving to asecond position within aperture 203. The time duration between t₀ and t₁corresponds to an amount of time that reporter region 214 spent at thefirst position. At time t₂, the value (e.g., magnitude) of signal canchange to a third value, corresponding to reporter region 214translationally, rotationally, or conformationally moving to a thirdposition. The time duration between t₁ and t₂ corresponds to an amountof time that reporter region 214 spent at the second position beforemoving to the third position. At time t₃, the value (e.g., magnitude) ofsignal can change to the first value, corresponding to reporter region214 translationally, rotationally, or conformationally returning to thefirst position. The time duration between t₂ and t₃ corresponds to anamount of time that reporter region 214 spent at the third positionbefore returning to the first position. It should be appreciated thatthe particular values and time periods of the signals illustrated inFIG. 2B are intended to be purely exemplary, and not limiting in anyway.

In one illustrative embodiment, reporter region 214 includes anelectrostatic charge, and the signal generated by system 220 includesthe current or flux through constriction 204 or optical signal. However,it should be understood that measurement circuit 230 can include, or bein communication with, any element or combination of elements thatfacilitates measurement of any suitable reporter region, and need notnecessarily be based on the measurement of current or flux throughconstriction 204 or an optical signal, or even based on the movement ofthe reporter region. Additionally, the reporter region 214 need notnecessarily be attached to the tether, and instead can be attached to anucleotide or other molecule being acted upon. The particular propertiesof the reporter region can be selected based on the particularconfiguration of measurement circuit 230 so as to facilitate measurementof that reporter region. For example, the reporter region can have anoptical property, and measurement circuit 230 can include, or be incommunication with, an optical sensor configured to measure the opticalproperty and to generate a signal based on the presence of or movementof the reporter region. In one illustrative embodiment, the reporterregion can include a first FRET pair partner, e.g., a FRET donor oracceptor, that interacts with a corresponding second FRET pair partner,e.g., a FRET acceptor or donor, so as to emit light of a particularwavelength that measurement circuit 230 is configured to detect. Or, forexample, the reporter region can have a chemical or biological property,and measurement circuit 230 can include, or be in communication with, achemical or biological sensor configured to measure the chemical orbiological property and that generates a signal based on the presence ofor movement of the reporter region. As another example, the reporterregion can provide a molecular flux blockade that modulates the flux ofmolecules through the aperture or constriction, which flux can bedetected optically, electrically, chemically, or biologically.

In one exemplary embodiment, reporter region 214 can be translationallymovable toward first side 201 of nanopore 200 responsive to a firstevent. The first event can be individually identifiable based on ameasured magnitude or time duration, or both, of a signal (e.g., anoptical or electrical signal) generated by system 220. For example, thefirst event can cause reporter region 214 to translationally move to afirst location, and the presence of reporter region 214 at the firstlocation causes the signal to have a first magnitude. As such, thesignal having the first magnitude correlates to the first event havingoccurred. Or, for example, the first event can cause reporter region totranslationally move to the first location for a first period of time,and the presence of reporter region 214 at the first location causes thesignal to have a first time duration. As such, the signal having thefirst time duration correlates to the first event having occurred. Inone specific example, the signal has both a first magnitude and a firsttime duration, each of which is based on the presence of reporter region214 at the first location, thus increasing confidence based on thesignal in a determination that a conformation change has occurred.Reporter region 214 can remain at the first location following the firstevent. Alternatively, reporter region 214 can be movable toward secondside 202 of nanopore 200 after the first event. For example, reporterregion 214 can return to a previous location, or to a differentlocation, after the first event.

Additionally, in some embodiments, reporter region 214 also can bemovable toward first side 201 of nanopore 200 responsive to a secondevent that occurs after the first event. The second event can beindividually identifiable based on a measured magnitude or timeduration, or both, of a signal (e.g., an optical or electrical signal)generated by system 220. For example, the second event can causereporter region 214 to move to a second location, and the presence ofreporter region 214 at the second location causes the signal to have asecond magnitude. As such, the signal having the second magnitudecorrelates to the second event having occurred. Or, for example, thesecond event can cause reporter region to move to the second locationfor a second period of time, and the presence of reporter region 214 atthe second location causes the signal to have a second time duration. Assuch, the signal having the second time duration correlates to thesecond event having occurred. In one specific example, the signal hasboth a second magnitude and a second time duration, each of which isbased on the presence of reporter region 214 at the second location,thus increasing confidence in a determination based on the signal that aconformation change has occurred. Reporter region 214 can remain at thesecond location following the second event. Alternatively, reporterregion 214 can be movable toward second side 202 of nanopore 200 afterthe second event. For example, reporter region 214 can return to anoriginal location, or to a different location, after the second event.The first and second events can be individually identifiable anddistinguishable from one another based on respective measuredmagnitudes, or time durations, or both, of the signals (e.g., optical orelectrical signals) generated by system 220.

In one non-limiting example, conformational motion can be measured. Forexample, it is well known that the distance between bases in extendedsingle stranded DNA (ssDNA) can be greater than that in double strandedDNA (dsDNA). For example, a ssDNA tether that is anchored adjacent tothe first side of a biological MspA nanopore and is stretched due to anapplied electric field can have inter-base distances of about 4.9Angstroms per base. Double stranded DNA (dsDNA), on the other hand, hasa spacing of about 3.32 Angstroms per base. Tether 211 can include anarbitrary DNA sequence and can be permanently anchored to MspA nanopore200 such that under the applied force created by the electric field,reporter region 214 that includes one or more abasic residues isdisposed at the main MspA constriction. The abasic reporter residue(s)214 can be flanked by deoxythymidine residues which have a verydifferent blockage current or flux in the MspA pore than the abasicsite(s). A conformational change can be induced in the elongated body213 of the tether 211 by hybridization of a complementaryoligonucleotide to the tether. The conformational change results fromthe conversion of ssDNA to dsDNA due to the inter-base spacingdifferences between ssDNA and dsDNA. The conformational change occurswithin the elongated body 213 which results in movement of the reporter214 out of the constriction 204 towards the first side, and the movementof deoxythymidine residues into the constriction zone. Due to thedifferent blockage currents or fluxes of these moieties, a change incurrent or flux signal occurs, which can be readily detected, e.g.,electrically or optically. For further details regarding interactionsbetween MspA and ssDNA, see Manrao et al., “Nucleotide Discriminationwith DNA Immobilized in the MspA Nanopore,” PLos ONE 6: e25723, 7 pages,(2011), the entire contents of which are incorporated by referenceherein.

Note that the location of reporter region 214, and the resulting signalgenerated by system 220, need not necessarily be responsive solely tooccurrence of an event, but can be responsive to any suitable stimulus.For example, measurement circuit 230 can be configured to apply avoltage between first electrode 231 and second electrode 232 so as toapply a voltage across nanopore 200, which causes reporter region 214 totranslationally move towards a given location, e.g., towards second side202 of nanopore 200. The occurrence of the event prior to or during suchmotion can define a location at which reporter region 214 stops (even iftransiently), which can define the signal (e.g., an optical orelectrical signal) that system 220 generates. Additionally, note that inembodiments such as described above with reference to FIG. 1M, in whichhead region 211 of tether 210 is attached to a first member and tailregion 212 of tether 210 is attached to a second member, neither ofwhich members need be attached to the first or second side of thenanopore, applying a voltage between first electrode 231 and secondelectrode 232 can cause a corresponding net movement of the tether 210and the first and second members either towards first electrode 231 ortowards second electrode 232. Optionally, one or both of the first andsecond members is sufficiently large as to be unable to pass fullythrough aperture 203 of nanopore 200. For example, based uponapplication of a first appropriate voltage between first electrode 231and second electrode 232, tether 210 and the first and second membersattached thereto can move towards first electrode 231, which movementcan cause the first member (attached to head region 211 in a manneranalogous to that illustrated in FIG. 1M) to become temporarily lodgedin a first location relative to aperture 203, e.g., disposed adjacent toaperture 203 on first side 201 or fully or partially disposed withinaperture 203 on first side 201 without passing fully through aperture203, thus inhibiting further movement of tether 210 and the first andsecond members towards first electrode 231. Or, for example, based uponapplication of a second appropriate voltage between first electrode 231and second electrode 232, tether 210 and the first and second membersattached thereto can move towards second electrode 232, which movementcan cause the second member (attached to tail region 212 in a manneranalogous to that illustrated in FIG. 1M) to become temporarily lodgedin a second location relative to aperture 203, e.g., disposed adjacentto aperture 203 on second side 202 or fully or partially disposed withinaperture 203 without passing fully through aperture 203, thus inhibitingfurther movement of tether 210 and the first and second members towardssecond electrode 232. As such, even if alternating voltages are appliedacross first electrode 231 and second electrode 232, tether 210 and thefirst and second members can be retained relative to nanopore 200.

In embodiments that include optional constriction 204, based on therelative width of reporter region 214, the length of elongated body 213,and the diameter of constriction 204 (e.g., dimension D2 illustrated inFIG. 1A), reporter region 214 can in some embodiments be movableadjacent to, into, or even through constriction 204, e.g., responsive toan event or other stimulus. For example, reporter region 214 can bedisposed within constriction 204, and then pulled out of constriction204 toward first side 201 responsive to the event or other stimulus. Or,for example, reporter region 214 can be disposed on second side 202 ofnanopore 200, and then pulled through constriction 204 and onto firstside 201 of nanopore 200 responsive to the event or other stimulus.

Additionally, note that system 220 suitably can be configured so as togenerate signals (e.g., optical or electrical signals) based uponreporter regions that are disposed on members other than on permanenttether 210. For example, tether 210 can interact with another moleculeto which a reporter region is attached. Such an interaction can causethe reporter region of the other molecule to move to a location, and thepresence of the reporter region at that location can cause the signalgenerated by system 220 to have a magnitude, or time duration, or bothmagnitude and time duration, that correlates to the interaction havingoccurred. For example, an interaction between tether 210 and anothermolecule can cause a reporter region attached to that molecule to becomepositioned at a location at which the reporter region is detectable bycircuit 230.

It further should be appreciated that an array of nanopores can beprovided so as to detect a plurality of events occurring in parallelwith one another. For example, FIG. 2C schematically illustrates a planview of a system 260 including measurement circuitry 240 configured tomeasure movement of respective reporter regions within the respectiveapertures of an array of nanopores. A plurality of systems 250, whichcan be configured analogously to system 220 described above withreference to FIGS. 2A-2B, can be integrally disposed in a commonsubstrate as one another, or can be separately prepared and disposedadjacent to one another. Each system 250 can include nanopore 200, atether (tether not specifically illustrated), and an addressableelectrode 241. Measurement circuit 240 can be configured analogously tomeasurement circuit 230, can be in electrical communication with eachaddressable electrode 241 of each system via a suitable communicationpath, e.g., conductor (communication illustrated for only a singlesystem 250) and with a common electrode 242. Measurement circuit 240 canbe configured to selectably apply a voltage across each nanopores 200 byapplying a voltage across the addressable electrode 241 of that nanoporeand across common electrode 242, and to selectably measure a current orflux through that nanopore or an optical signal at the applied voltage.An event can be detected based on such a current or flux or opticalsignal, e.g., such as described elsewhere herein. Analogous arraysreadily can be envisioned for other types of detection systems, e.g.,light, chemical, or biological detection systems.

Exemplary Methods and Exemplary Compositions for Use During Such Methods

Some exemplary methods for detecting events, and exemplary compositionsthat can be used during such methods, now will be described. Under oneaspect, a method includes providing a nanopore including a first side, asecond side, and an aperture extending through the first and secondsides; and providing a permanent tether including a head region, a tailregion, and an elongated body disposed therebetween. The head region canbe anchored to or adjacent to the first or second side of the nanopore,and the elongated body can include a reporter region. The method caninclude moving the reporter within the aperture responsive to a firstevent occurring adjacent to the first side of the nanopore. In someembodiments, the reporter region is translationally moved within theaperture responsive to the first event. Additionally, or alternatively,the reporter region can be rotationally moved within the apertureresponsive to the first event. Additionally, or alternatively, thereporter region is conformationally moved within the aperture responsiveto the first event.

For example, FIG. 3A illustrates an illustrative method 300 fordetecting an event using a composition including a tether anchored to oradjacent to a nanopore. Method 300 includes providing a nanoporeincluding a first side, a second side, and an aperture extending throughthe first and second sides (step 301). The nanopore can have anysuitable configuration, e.g., such as described above with reference toFIGS. 1A-1M. For example, nanopore 100 illustrated in FIG. 1A includesfirst side 101, second side 102, and aperture 103 extending through thefirst and second sides. Or, for example, nanopore 100′ illustrated inFIG. 1J includes first side 101′, second side 102′, an aperture definedby aperture 103′ and constriction 104′. Or, for example, nanopore 100″illustrated in FIG. 1K includes first side 101″, second side 102″, andaperture 103″ extending through the first and second sides, e.g.,defined by biological pore 108″. Or, for example, nanopore 100′″illustrated in FIG. 1L includes first side 101′″, second side 102′″, andaperture 103′″ extending through the first and second sides, e.g.,defined through layer 107′″.

Step 301 also can, but need not necessarily, include preparing thenanopore. For example, step 301 can include defining a barrier anddisposing a nanopore on or in the barrier. Methods of preparingnanopores are known in the art. For example, illustrative methods ofpreparing an MspA nanopore can be found in Butler et al.,“Single-molecule DNA detection with an engineered MspA proteinnanopore,” Proc. Natl. Acad. Sci. 105: 20647-20652 (2008), the entirecontents of which are incorporated by reference herein. Or, for example,illustrative methods of preparing an alpha hemolysin nanopore can befound in Howorka et al., “Sequence-specific detection of individual DNAstrands using engineered nanopores,” Nature Biotechnology 19: 636-639(2001), and in Clarke et al., “Continuous base identification forsingle-molecule nanopore DNA sequencing,” Nature Nanotechnology 4:265-270 (2009), the entire contents of both of which are incorporated byreference herein.

Method 300 illustrated in FIG. 3A also includes providing a permanenttether including a head region, a tail region, and an elongated bodytherebetween, the elongated body including a reporter region, the headregion being anchored to or adjacent to the first side or second side ofthe nanopore (step 302). The tether can have any suitable configuration,such as described above with reference to FIGS. 1A-1M. For example, theelongated body can be of a length that is shorter than a first dimensionH1 defining a thickness of the nanopore, e.g., such as illustrated inFIGS. 1A, 1B, and 1J. Or, for example, the elongated body can be of alength that is longer than a first dimension H1 defining a thickness ofthe nanopore, e.g., such as illustrated in FIGS. 1G and 1K. Or, forexample, in embodiments that include a constriction, the elongated bodycan be of a length that is shorter than a second dimension H2 defining aconstriction depth, e.g., such as illustrated in FIG. 1A. Or, forexample, in embodiments that include a constriction, the elongated bodycan be of a length that is longer than a second dimension H2 defining aconstriction depth, e.g., such as illustrated in FIGS. 1B, 1G, 1J, and1K. Or, for example, the reporter region can be disposed at a locationalong the elongated body that is selected such that, based upon theelongated body being fully or partially extended when the head region isanchored to or adjacent to the nanopore, the reporter region ispositionable within the aperture of the nanopore, e.g., adjacent to orwithin an optional constriction, such as illustrated in FIGS. 1C, 1J,and 1K. Any suitable combination of such features can be used.

Step 302 also can, but need not necessarily, include preparing thetether. For example, step 302 can include defining an elongated bodythat includes portions thereof defining a head region, tail region, andone or more reporter region(s). For example, as described elsewhereherein, a tether can include DNA. A DNA oligonucleotide of sufficientlength can be prepared using procedures well known in the art. Forexample, oligonucleotides with a 5′ or 3′ primary amine can be purchasedcommercially from vendors such as Integrated DNA Technologies, Inc.(Coralville, Iowa). The oligonucleotide can be ordered so as to includeone or more abasic moieties, which can be used as one or more reporterregions as described herein. A bifunctional linker, such as sulfo-SMCC(sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) thatincludes an amine reactive group (NHS) and a thiol reaction group(maleimide) can be readily obtained from commercial sources, e.g., fromThermo Fisher Scientific, Inc. (Rockford, Ill.). Such a linker can bereacted with the oligonucleotide under appropriate reaction conditionswell known in the art to form a stable amide bond. After purification ofthe oligonucleotide from the unreacted sulfo-SMCC, the modifiedoligonucleotide (which is now thiol reactive by virtue of its maleimidegroup) can be reacted with the nanopore, e.g., protein nanopore. Theprotein nanopore can be prepared in advance so as to include at leastone solvent accessible cysteine residue that has its thiol (SH) group inreduced form. The reduced form can be obtained by incubation with 5 mMtris(2-carboxyethyl)phosphine (TCEP), for example, which is a readilyavailable commercial compound. The modified oligonucleotide can becombined, e.g., in molar excess, with the reduced protein nanopore andunder reaction conditions well known in the art, such that the maleimideforms a stable thioether bond. The protein-oligonucleotide conjugate canbe purified away from excess unreacted oligonucleotide. In anotherexample, compounds suitable for inclusion in a polyethylene glycol (PEG)based tether, e.g., maleimide-PEG, are readily available from commercialsources, such as Laysan Bio, Inc. (Arab, Ala.). For example, themaleimide can be conjugated to a reduced cysteine thiol in a manneranalogous to that described above. A suitable reporter region can bedefined within the PEG. In another example, a disulfide bond between anoligonucleotide and an alpha hemolysin nanopore can be prepared in amanner such as described in Howorka et al., “Kinetics of duplexformation for individual DNA strands within a single protein nanopore,”PNAS 98: 12996-13301 (2001), the entire contents of which areincorporated by reference herein.

Additionally, or alternatively, step 302 optionally can includeanchoring the head region of the tether to or adjacent to the first sideor the second side of the nanopore. For example, the head region of thetether can be attached to or adjacent to the first side or the secondside of the nanopore using a chemical bond, e.g., a covalent bond,hydrogen bond, ionic bond, dipole-dipole bond, London dispersion forces,or any suitable combination thereof. Or, for example, the head region ofthe tether can be attached to the first side or the second side of thenanopore using an interaction between a first protein structure on thehead region and a second protein structure that is attached to, oradjacent to, the first or second side of the nanopore. For example, thefirst and second structures can include alpha helices that interlockwith one another. The attachment of the head region of the tether to oradjacent to the first or second side of the nanopore can be permanent,such that the head group of the tether is held in a generally fixedposition with respect to the first or second side of the nanopore. Forexample, the head region can be anchored to the first side of thenanopore, e.g., as illustrated in FIGS. 1A, 1J, and 1K. Or, for example,the head region can be anchored to the second side of the nanopore,e.g., as illustrated in FIG. 1D. Or, for example, the head region can beanchored adjacent to the first side of the nanopore, e.g., anchored to amember that is disposed adjacent to, and optionally is attached to, thefirst side of the nanopore such as illustrated in FIGS. 1E, 1F, and 1M.Note that even if such member moves translationally or conformationallyadjacent to the nanopore, the tether anchored thereto still can beconsidered to be anchored adjacent to the nanopore. Analogously, thehead region can be anchored adjacent to the second side of the nanoporeor to another member that is disposed adjacent to, and optionally isattached to, the second side of the nanopore (not specificallyillustrated).

In one illustrative embodiment, the reduced thiol (—SH) group (alsocalled a sulfhydryl group) of a cysteine residue can be reacted with atether having a thiol-reactive group. Examples of such groups includemaleimide and iodoacetamide. As described in greater detail atwww.lifetechnologies.com/us/en/home/references/molecular-probes-the-handbook/thiol-reactive-probes/introduction-to-thiol-modification-and-detection.html#head2,primary thiol-reactive reagents, including iodoacetamides, maleimides,benzylic halides, and bromomethylketones can react by S-alkylation ofthiols so as to generate stable thioether products; arylating reagentssuch as 7-nitrobenz-2,1,3-oxadiazole (NBD) halides can react with thiolsor amines by a similar substitution of the aromatic halide by thenucleophile; and because the thiolate anion is a better nucleophile thanthe neutral thiol, cysteine is more reactive above its pKa.Additionally, as described in greater detail atwww.piercenet.com/method/sulfhydryl-reactive-crosslinker-chemistry,sulfhydryl-reactive chemical groups include haloacetyls, maleimides,aziridines, acryloyls, arylating agents, vinylsulfones, pyridyldisulfides, TNB-thiols (2-nitro-5-thiobenzoic acid), and disulfidereducing agents; such groups can conjugate to sulfhydryls via alkylation(e.g., via formation of a thioether bond) or disulfide exchange (e.g.,formation of a disulfide bond). Sulfhydryl exchange reactions alsosuitably can be used. Alternatively, Amines (—NH₂) can be targeted. Forexample, the primary amine of the lysine residue and the polypeptideN-terminus are relatively reactive. Amine residues can be targeted withN-hydroxysuccinimide esters (NHS esters), which can form a stable amidebond, or imidoester crosslinkers, which can react with primary amines toform amidine bonds. There are many other amine-reactive compounds. Forexample, as described atwww.piercenet.com/method/amine-reactive-crosslinker-chemistry, syntheticchemical groups that can form chemical bonds with primary amines includeisothiocyanates, isocyanates, acyl azides, NHS esters, sulfonylchlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, arylhalides, imidoesters, carbodiimides, anhydrides, and fluorophenylesters; such groups can conjugate to amines, for example, via acylationor alkylation. In still other embodiments, a modified amino acid residuecan be used to introduce a novel functionality like an azide or alkyneto be used with click chemistry. For example, thiol or aminereactivities such as described above can be used with linkers thatpermit the addition of azide or alkyne functionalities to further beused in a click chemistry reaction.

In the embodiment illustrated in FIG. 3A, method 300 includes moving thereporter region within the aperture of the nanopore responsive to anevent occurring adjacent to the first side of the nanopore (step 303).Such movement can be translational, rotational, or conformational, orany suitable combination thereof. For example, the event can causetranslational movement of the head region toward the first side of thenanopore, and the translational movement of the head region can causemovement of the elongated body, or a portion thereof, and the reporterregion toward the first side of the nanopore. Or, for example, the eventcan cause translational movement of a portion of the elongated bodytoward the first side of the nanopore, and the translational movement ofthe portion of the elongated body can cause translational movement ofthe reporter region toward the first side of the nanopore. In oneillustrative embodiment, the reporter region initially is disposed in,or adjacent to, a constriction of the nanopore, the event causes thereporter region to move away from the constriction towards the firstside, to a first location.

Optionally, method 300 also includes moving the reporter region towardthe second side of the nanopore after the event (not specificallyillustrated). For example, after the event, the head region cantranslationally move toward the second side of the nanopore, and themovement of the head region can cause translational movement of theelongated body, or a portion thereof, and the reporter region toward thesecond side of the nanopore. Or, for example, after the event, a portionof the elongated body can translationally move toward the second side ofthe nanopore, and the movement of the portion of the elongated body cancause translational movement of the reporter region toward the secondside of the nanopore. Or, for example, a stimulus, such as an appliedvoltage, can cause translational movement of the reporter region towardthe second side of the nanopore. In one illustrative embodiment, afterthe event, the reporter region translationally moves from a firstlocation to which it had moved responsive to the first event, towardsthe second side and towards the constriction, and optionallytranslationally moves adjacent to or into the constriction. As notedabove, the reporter region can be repeatedly movable, e.g.,translationally, rotationally, or conformationally, within the apertureresponsive to different events, thus facilitating detection of each suchevent.

It should be appreciated that step 302 can be performed, or any othercomposition provided herein can be prepared, using any suitablecombination of steps. For example, FIG. 3B illustrates a method forpreparing a composition including a tether and a polymerase adjacent toa nanopore, according to some embodiments of the present invention.Method 310 includes attaching a head region of a tether to one of afirst side or a second side of a nanopore or to a first member (311).For example, method 310 can include attaching the head region of thetether directly to the first side or second side of a nanopore, in amanner analogous to that described above with reference to FIG. 1A-1C,1D, or 1G-1L using any suitable attachment provided herein or otherwiseknown in the art. Or, for example, method 310 can include attaching thehead region of the tether directly to a first member in a manneranalogous to that illustrated in FIG. 1E, 1F, or 1M using any suitableattachment provided herein or otherwise known in the art.

In embodiments in which the head region of the tether is attached to afirst member, method 310 illustrated in FIG. 3B optionally can includeattaching the first member to one of the first side or the second sideof the nanopore (312). For example, a first member having a head regionof a tether attached thereto, such as described above with reference toFIG. 1E, 1F, or 1M, can be attached to the first side or the second sideof the nanopore. Alternatively, a first member having a head region of atether attached thereto, such as described above with reference to FIG.1E, 1F, or 1M, can be disposed adjacent to the first side or the secondside of the nanopore without attaching the first member thereto.

Method 310 illustrated in FIG. 3B further can include disposing theelongated body of a tether within an aperture of the nanopore (313).Based on the length of the elongated body, the elongated body can, butneed not necessarily, extend all the way through the aperture of thenanopore. For example, in embodiments such as described above withreference to FIGS. 1A, and 1E, the elongated body of the tetheroptionally can be sufficiently short that the tail region of the tetherremains on the same side of a constriction (if present) of the nanoporeas is the head region of the tether. Or, for example, in embodimentssuch as described above with reference to FIG. 1B-1D, 1F, 1J, or 1L, theelongated body of the tether optionally can be sufficiently long thatthe tail region of the tether remains disposed within the aperture ofthe nanopore, and optionally can be sufficiently long that the tailregion of the tether is disposed on the other side of a constriction (ifpresent) of the nanopore as is the head region of the tether. Or, forexample, in embodiments such as described above with reference to FIG.1G-1I, 1K, or 1M, the elongated body of the tether optionally can besufficiently long that the tail region of the tether can be disposedbeyond the other side of the nanopore as is the head region of thetether.

Illustratively, the elongated body of the tether can be disposed withinthe aperture of the nanopore by applying a suitable directional force tothe elongated body of the tether. For example, a voltage can be appliedacross the nanopore in a manner such as described herein with referenceto FIGS. 2A-2C, and the elongated body of the tether can include atleast one charged moiety that, based on the voltage, attracts the tailregion of the tether towards the side of the nanopore opposite that towhich the head region of the tether is attached or at which the headregion of the tether is attached to a first member, and causestranslocation of the tail region so as to dispose all or a portion ofthe elongated body of the tether within the aperture of the nanopore.Note that in embodiments in which the head region of the tether isattached to a first member, such a directional force also can bring thefirst member adjacent to, or fully or partially disposed within, theaperture of the nanopore in a manner such as described herein withreference to FIG. 1M. For example, the attraction of the tail regiontowards the side of the nanopore opposite that at which the head regionof the tether is attached to a first member also can cause translocationof the first member to a position adjacent to, or fully or partiallydisposed within, the aperture of the nanopore.

Method 310 illustrated in FIG. 3B optionally further can includeattaching the tail region of the tether to the other of the first sideor second side of the nanopore or to a second member (314). Any suitableattachment such as provided herein, or otherwise known in the art,suitably can be used. For example, method 310 optionally can includeattaching the tail region of the tether to the side of the nanoporeopposite of that of the head region, such as described above withreference to FIG. 1H. Or, for example, method 310 optionally can includeattaching the tail region of the tether to a second member disposed onthe side of the nanopore opposite to that of the head region, such asdescribed above with reference to FIG. 1I or 1M. The second memberoptionally can be disposed within the aperture of the nanopore.Alternatively, step 314 need not be performed, and method 310 caninclude allowing the tail region of the tether to extend freely withinthe aperture of the nanopore, in a manner such as described above withreference to FIG. 1A-1F, 1J, or 1L, or beyond the aperture of thenanopore, in a manner such as described above with reference to FIG. 1Gor 1K.

Under some conditions, the application of a directional force to theelongated body of a tether can cause translocation of the tail region soas to dispose all of the tether within the aperture of the nanopore. Asufficiently large force can cause a polymerase (or other protein) thatis attached to the tether to become temporarily lodged in or on thenanopore. Although not intending to be a limiting with respect tophysical configuration, the result can be termed ‘corking’ of thenanopore by the protein. Corking can be inhibited or avoided by limitingthe force on the tether (e.g., applying less than 180 mV across thenanopore), limiting the duration of time that force is applied on thesystem, or using a sufficiently large protein that the corkinginteraction is avoided. Alternatively or additionally, a reverse voltagecan be applied to the system to reverse the interaction between theprotein and nanopore (referred to as ‘uncorking’). Another option toinhibit or avoid corking or to facilitate uncorking is to remove chargedamino acids from the nanopore opening or complementary charges on thesurface of the protein, so as to reduce charge affinity between the twocomponents. It can be further beneficial to add cross links to thestructure of the protein (e.g., engineered cysteine pairs that fordisulfide crosslinks or chemical crosslinkers), in order to stabilizethe globular structure of the protein.

Corking can be observed based on a characteristic current or flux oroptical pattern that is distinct from patterns resulting from otherconfigurations of the nanopore system. The distinct pattern can beobserved for example, when applying a negative bias to the nanoporesystem. Accordingly, current or flux or optical patterns can be detectedduring assembly or use of a system that includes a protein that islocalized to a nanopore via tether that is attached to the protein anddisposed in the nanopore lumen. Detection of the patterns can be used tomonitor assembly (e.g., to avoid corking), guide uncorking, or otherwiseoptimize desired assembly.

It should be appreciated that the present compositions, systems, andmethods suitably can be used to detect many types of events. Forexample, the present compositions, systems, and methods suitably can beused to detect the motion of a molecule or a portion of that molecule.In one illustrative embodiment, the motion includes a conformationalchange of the molecule. In another illustrative embodiment, the motionincludes an interaction of a molecule with another molecule, such as afirst molecule binding another molecule, e.g., a protein binding anucleotide, or a nucleotide being added to a polynucleotide. Otherevents can be envisioned.

Exemplary Methods and Compositions for Detecting Conformational Changesof Molecules

FIG. 4A illustrates exemplary method 400 for detecting a conformationalchange of a molecule using a composition including a tether anchored toor adjacent to a nanopore. It should be appreciated that method 400suitably can be adapted to detecting conformational changes of manytypes of molecules, such as proteins and nucleic acids.

Method 400 illustrated in FIG. 4A includes providing a compositionincluding a nanopore, a permanent tether, and a molecule disposedadjacent to the nanopore (step 401). A composition can include ananopore including a first side, a second side, and an apertureextending through the first and second sides; and a permanent tetherincluding a head region, a tail region, and an elongated body disposedtherebetween. The head region can be anchored to or adjacent to thefirst side or second side of the nanopore. The elongated body includinga reporter region can be movable within the aperture responsive to afirst event occurring adjacent to the first side of the nanopore. Forexample, FIGS. 5A-5B schematically illustrate a composition including atether anchored adjacent to a nanopore and configured for use indetecting a conformational change of a molecule disposed adjacent to thenanopore. In the exemplary embodiment illustrated in FIG. 5A, thecomposition can include nanopore 500, permanent tether 510, and molecule550. Nanopore 500 includes first side 501, second side 502, aperture503, and optionally also includes constriction 504. Permanent tether 510includes head region 511, tail region 512, and elongated body 513disposed therebetween and including reporter region 514 (optionally, oneor more additional reporter regions can be provided such as describedabove with reference to FIG. 1C).

Molecule 550 can be disposed adjacent to first side 501 of nanopore 500.For example, molecule 550 can be in contact with first side 501 ofnanopore 500, and optionally can be anchored to or adjacent to the firstside of nanopore 500 via any suitable chemical bond, protein-proteininteraction, or any other suitable attachment that is normallyirreversible. In one illustrative embodiment, molecule 550 includes aprotein. One example of a protein suitable for use in method 400illustrated in FIG. 4A is an enzyme. One example of an enzyme suitablefor use in method 400 illustrated in FIG. 4A is a polymerase. Othertypes of molecules, proteins, or enzymes suitably can be used. In theembodiment illustrated in FIG. 5A, head region 511 of tether 510 isattached to, e.g., anchored to, molecule 550, via any suitable chemicalbond, protein-protein interaction, or any other suitable attachment thatis permanent. Head region 511 can be attached to any suitable portion ofmolecule 550 that undergoes a conformational change that can causemovement of reporter region 514 relative to constriction 504. Note thatmolecule 550 need not necessarily be considered to be part of theinventive composition, but instead can be considered to be in contactwith a composition that includes nanopore 500 and permanent tether 510.Additionally, note that molecule 550 can be, but need not necessarilybe, attached to or adjacent to nanopore 500. Additionally, note thattail region 512 optionally can be attached to a second molecule (notspecifically illustrated) in a manner such as described above withreference to FIGS. 1I and 1M.

Referring again to FIG. 4A, method 400 includes changing theconformation of the molecule (step 402). For example, FIG. 5Bschematically illustrates a conformational change to molecule 550 thatcauses motion of one or more regions of molecule 550 relative to one ormore other regions of molecule 550. In one illustrative embodiment inwhich molecule 550 is a polymerase, the conformational change of thepolymerase can be responsive to the polymerase binding a nucleotide.Alternatively, the conformational change of the polymerase can beresponsive to the polymerase adding a nucleotide to a polynucleotide. Instill other alternative embodiments, the conformational change of thepolymerase can be responsive to the polymerase binding to a nucleic acidtemplate, releasing a nucleic acid template, releasing a nucleotidewithout incorporating it, or excising a nucleotide, or a combinationthereof.

Referring again to FIG. 4A, method 400 also includes translationallymoving the head region of the tether responsive to the conformationalchange of the molecule (step 403). For example, FIG. 5B schematicallyillustrates a conformational change to molecule 550 that moves headregion 511. Such movement of region can be, but need not necessarily be,away from nanopore 500. For example, in the embodiment illustrated inFIG. 5B, head region 511 is moved both laterally relative to aperture503 and away from first side 501 of nanopore 500.

Referring again to FIG. 4A, method 400 also includes translationallymoving the reporter region within the nanopore aperture responsive tomovement of the head region (step 404). For example, FIG. 5Bschematically illustrates a conformational change to molecule 550 thattranslationally moves head region 511, and the movement of head region511 translationally moves reporter region 514 toward first side 501, asindicated by the dashed arrow. For example, reporter region 514 can beadjacent to or disposed within optional constriction 504 prior to theconformational change, such as illustrated in FIG. 5A, and can be movedaway from optional constriction 504 toward first side 501 responsive tothe conformational change. Alternatively, a conformational change tomolecule 550 instead can move head region 511 in such a manner thatreporter region 514 translationally moves toward second side 502, orundergoes any other suitable translational, rotational, orconformational movement, or a combination thereof, within aperture 503.

Referring back to FIG. 4A, method 400 further includes detecting themovement of the reporter region (step 405). For example, the compositioncan be in operable communication with a measurement circuit such asdescribed above with reference to FIG. 2A or FIG. 2C. The measurementcircuit can be configured to detect the movement of the reporter regionwithin the aperture. In one illustrative embodiment, nanopore 500,tether 510, and molecule 550 can be immersed in a conductive fluid,e.g., an aqueous salt solution. A measurement circuit configuredanalogously to measurement circuit 230 illustrated in FIG. 2A ormeasurement circuit 240 illustrated in FIG. 2C can be in communicationwith first and second electrodes and can be configured to apply avoltage between those electrodes so as to impose a voltage acrossnanopore 500. The measurement circuit further can be configured to usethe electrodes to measure the magnitude of a current or flux throughaperture 503 or can include an optical sensor to measure an opticalsignal. Reporter region 514 can have a different current or fluxblockade property, e.g., a different physical, chemical, biological,optical, or electrical property, than some or all other regions ofelongated body 513. For example, reporter region 514 can include anelectrostatic charge, while some or all other regions of elongated body513 can include a different electrostatic charge, or can be uncharged(e.g., can be electrically neutral). Or, for example, reporter regioncan be uncharged, while some or all other regions of elongated body 513can include an electrostatic charge. The magnitude of the current orflux through aperture 503 or optical signal can measurably changeresponsive to a change in the position of reporter region 214 relativeto constriction 204, and the time period for such a measurable change inthe current or flux or optical signal is based on the duration of thereporter region's change in position. In one illustrative, nonlimitingexample, elongated body 513 includes a polynucleotide that includes oneor more abasic nucleotides that define reporter region 514.

The change in conformation of molecule 550 can be individuallyidentifiable based on a measured (e.g., optically or electricallymeasured) magnitude or time duration, or both, of a signal generated bysuch a system. For example, the conformational change can cause reporterregion 514 to move to a first location, and the presence of reporterregion 514 at the first location causes the signal (e.g., an optical orelectrical signal) to have a first magnitude. As such, the signal havingthe first magnitude correlates to the conformation change havingoccurred. Or, for example, the conformation change can cause reporterregion 514 to move to the first location for a first period of time, andthe presence of reporter region 514 at the first location causes thesignal to have a first time duration. As such, the signal having thefirst time duration correlates to the conformation change havingoccurred. In one specific example, the signal has both a first magnitudeand a first time duration, each of which is based on the presence ofreporter region 514 at the first location, thus increasing confidence ina determination based on the signal that a conformation change hasoccurred.

As illustrated in FIG. 4A, method 400 further can include returning tothe prior conformation of the molecule (step 406). Method 400 furthercan include translationally moving the head region of the tetherresponsive to return of the molecule to the prior confirmation (step407). Method 400 further can include translationally moving the reporterregion of the tether within the nanopore aperture responsive to movementof the head region (step 408). For example, following the conformationalchange illustrated in FIG. 5B, molecule 550 can return to the molecule'sprevious conformation, e.g., such as illustrated in FIG. 5A. Such areturn can move head region 511 in such a manner that reporter region514 can translationally move toward second side 502, e.g., to a locationadjacent to or within constriction 504. Alternatively, rather thanreturning to the prior conformation, the molecule instead can change toa different conformation that is different than the prior conformation.Method 400 further can include detecting the movement of the reporterregion (step 409). Such detection can be performed analogously asdescribed above with reference to step 406.

It should be appreciated that method 400 illustrated in FIG. 4A suitablycan be adapted to detect events other than conformational changes, e.g.,to detect translational molecular motions, or combinations of differenttypes of molecular motions. Additionally, it should be appreciated thatmethod 400 illustrated in FIG. 4A suitably can be adapted to detect suchevents using any suitable combination of translational, rotational, orconformational changes of the reporter region of the tether.

Sequencing by Synthesis Using Exemplary Methods and Compositions Basedon Detecting Conformational Changes of a Polymerase

It should be appreciated that method 400 illustrated in FIG. 4A suitablycan be used to detect any of a variety of conformational changes. In onenonlimiting, illustrative embodiment described below with reference toFIGS. 6A-6D, method 400 can be used to detect the conformational changeof a polymerase associated with the polymerase acting upon a nucleotide.Detection of such conformational changes can be used to sequence a firstpolynucleotide by synthesizing a second polynucleotide that iscomplementary to the first nucleotide, e.g., using “sequencing bysynthesis,” or SBS.

Previously known methods for SBS have been developed. For example,single stranded DNA (ssDNA) can pass through a biological nanopore, suchas a protein nanopore, that is embedded in a barrier such as a lipidbilayer, responsive to an electrical potential being applied across thenanopore. In what can be referred to as “strand” sequencing, asnucleotides of the ssDNA pass through a pore constriction, combinationsof those nucleotides can create unique current or flux blockadescorresponding to the identities of nucleotides in the particularcombinations pass through the constriction. These strands that are beingsequenced are not permanently attached to the pore or to the polymerase.Rather, these strands translocate through the pore such that the netposition of the strand changes relative to the pore. However, theextremely rapid translocation rate of ssDNA (˜1 nt/μsec), as well as thenative resolution of the constriction that encompasses a combination ofnucleotides, rather than a single nucleotide, can hinder accuratemeasurement of such current or flux blockades on anucleotide-by-nucleotide basis. Enzymatic “motors” have been used toslow the translocation speed to a rate which is more compatible withdata acquisition (milliseconds per nucleotide). However, such motorswhen used in strand sequencing configurations can introduce error modessuch as skipping, slipping and toggling, which can inhibit reliabledetection of nucleotides in the ssDNA. These and other motor-independenterror modes that can occur during strand sequencing can result from the“springiness” or elasticity of the ssDNA residing between the motor andthe constriction of the nanopore. Such springiness can be a function ofthe sequence of the ssDNA, and can result in different currents orfluxes for the same combination of nucleotides transiting theconstriction if different instances of that combination respectively aresurrounded by different ssDNA sequences. Further, because theconstriction can be relatively small, e.g., about 2 nt, and Brownianmotion is always present, the pore “read head” can be effectively about4 nucleotides in size, e.g., the constriction reads a combination ofabout 4 nucleotides at a time, thus making it more difficult to uniquelyidentify each nucleotide since there are 4̂4 (256) currents or fluxesthat need to be differentiated from one another.

Accordingly, a need remains for improvements in SBS, e.g., forinexpensive, accurate, long-read, high-throughput compositions, systems,and methods for SBS. SBS using biological nanopores represents onepotential solution to this need because of the nanoscale reproducibilityand ease of production of these proteins. Taken together, an approachwhich is motor free (e.g. using nucleic enzymes as a detector that iscoupled to a nanopore rather than as a motor that modulates passage of atarget strand through a nanopore), more tolerant of Brownian motion, andhas single nucleotide resolution can be expected to greatly advance thefield of nanopore DNA sequencing.

As noted above, the present methods, compositions, and systems can beused to detect conformational changes in a molecule. Accordingly, thepresent methods, compositions, and systems can be applied to monitoringconformational changes that a DNA polymerase undergoes as it synthesizesDNA from a template. For example, the polymerase can transition betweenwhat is referred to as an “open state,” in which the polymerase does notbind a nucleotide, to a “closed state,” in which the polymerase binds anucleotide. See, e.g., Xia et al., “Alteration in the cavity sizeadjacent to the active site of RB69 DNA polymerase changes itsconformational dynamics,” Nucl. Acids Res. (2013), nar.gkt674, theentire contents of which are incorporated by reference herein. See alsoSantoso et al., “Conformational transitions in DNA polymerase I revealedby single-molecule FRET,” Proc. Natl. Acad. Sci. USA, 107(2): 715-720(2010), the entire contents of which are incorporated herein byreference.

Conformational changes on the order of several nanometers are known tooccur during the catalytic cycle of nucleotide incorporation as thepolymerase transitions from the open to closed state, or as thepolymerase switches into editing mode. For example, FIGS. 6A-6Bschematically illustrate relatively large polymerase conformationchanges (>1 nm) in two different polymerases. FIG. 6A illustrates RB69polymerase, which exhibits a relatively large conformational change thatresults in relative movement between the thumb domain and finger domain,undergoing over 3 nm of movement between the open and closedconformations, as described by Xia et al. FIG. 6B illustrates Pol I(Klenow Fragment, or KF), which undergoes conformational changes duringnucleotide incorporation as disclosed by Santoso et al. The α-carbonbackbone of the polymerase is shown in beige. The DNA template strand isin dark gray, the primer strand in light gray. The terminal base pair atthe active site is magenta. According to Santoso, the 0 carbons of thetwo side chains were used as fluorophore attachment sites, shown asgreen and red spheres, to measure conformational changes of thepolymerase. The arrows indicate the distance in Angstroms between thegreen and red Cβ positions in the open and closed conformations. Therealso is evidence that the conformational changes of a polymerase can bedependent upon the identity of the nucleotide being incorporated, e.g.,such as described in Olsen et al., “Electronic Measurements ofSingle-Molecule Processing by DNA Polymerase I (Klenow Fragment),” JACS135: 7855-7860 (2013), the entire contents of which are incorporated byreference herein. In the nanopore embodiments of the present disclosure,the finger domain can be anchored to a nanopore while a tether isattached to the thumb domain. Alternatively, the thumb domain can beanchored to a nanopore while a tether is attached to the finger domain.In either construct, the relative movement that occurs between thefinger and thumb domains during polymerase activity can be detected asrelative movement between the tether and the nanopore. Attachmentchemistries used to attach optical probes (e.g., FRET pairs) in thereferences cited herein can be used in the nanopore embodiments setforth herein. Other attachment points can be used in apolymerase-nanopore construct so long as conformational changes in thepolymerase are reliably transmitted as relative movement between thetether and nanopore.

Using the present composition, a nanopore and a permanent tether can beused to transduce the conformational changes of a polymerase during SBSinto an electrical current or flux signature. Note that using thepresent compositions, systems, and methods, the DNA being sequencedduring SBS according to the present methods need not transit thenanopore, and can be sequenced on a nucleotide-by-nucleotide basis, thusdistinguishing the method from strand sequencing methods such asmentioned above and such as described in greater detail in U.S. PatentPublication No. 2014/0051096 to Jeyasinghe et al., the entire contentsof which are incorporated by reference herein.

More specifically, FIGS. 6C-6D schematically illustrate an exemplarycomposition including a permanent tether anchored to a polymerasedisposed adjacent to a nanopore and configured for use in detecting aconformational change of the polymerase responsive to binding of anucleotide. The nanopore includes biological pore 605, which can bedisposed in a barrier (not specifically illustrated), e.g., a membraneof biological origin such as a lipid bilayer, or a solid state membrane.Biological pore 605 includes aperture 603 and constriction 604. Thepermanent tether includes head region 611, tail region 612, elongatedbody 613, and reporter region 614. Optionally, tail region 612 can beattached to a second member (not specifically illustrated) in a manneranalogous as described with reference to FIGS. 1F and 1M. Polymerase 650is disposed adjacent to biological pore 605, and optionally can beattached to biological pore 605.

Polymerase 650 is configured to receive a template polynucleotide, e.g.,circular or linear ssDNA to be sequenced, to synthesize a polynucleotidehaving a complementary sequence to that of the ssDNA by sequentiallyreceiving, binding, and adding nucleotides to the polynucleotide inaccordance with the sequence of the ssDNA. Head region 611 of thepermanent tether is anchored to a location of polymerase 650 thatundergoes a conformational change, e.g., responsive to receiving anucleotide, binding a nucleotide, or adding a nucleotide topolynucleotide 660, and that moves reporter region 614 to a sufficientlydifferent location relative to constriction 604 so as to produce asignal from which an identity of that polynucleotide can be individuallydetermined. For example, head region 611 can be attached to a fingerregion of the polymerase, or a thumb of the polymerase. Exemplaryattachment points in the finger and thumb regions of polymerases andchemistries for attaching moieties to these points are set forth in U.S.Patent Publication No. 2011/0312529 A1, the entire contents of which areincorporated by reference herein. For further details on the structureand function of family A and B polymerases, see Patel et al., “Getting agrip on how DNA polymerases function,” Nature Structural Biology 8:656-659 (2001), the entire contents of which are incorporated byreference herein. For further details on the structure and function ofpolymerases such as Pol I, see the following references, the entirecontents of each of which are incorporated by reference herein: Olsen etal., “Electronic measurements of Single-Molecule Processing by DNAPolymerase I (Klenow Fragment,” JACS 135: 7855-7860 (2013); Torella etal., “Identifying molecular dynamics in single-molecule FRET experimentswith burst variance analysis,” Biophysics J. 100: 1568-1577 (2011);Santoso et al., “Conformational transitions in DNA polymerase I revealedby single-molecule FRET,” Proc. Natl. Acad. Sci. USA, 107(2): 715-720(2010), Markiewicz et al., “Single-molecule microscopy reveals newinsights into nucleotide selection by DNA polymerase I,” Nucleic AcidsRes. 40: 7975-7984 (2012); Gill et al., “DNA Polymerase activity at thesingle-molecule level,” Biochem. Soc. Trans. 39: 595-599 (2011), andJohnson et al., “Processive DNA synthesis observed in a polymerasecrystal suggests a mechanism for the prevention of frameshiftmutations,” Proc. Natl. Acad. Sci. USA 100: 3895-3900 (2003). Any tworesidues or domains that are known from the above references (or otherreferences cited herein) to undergo a change in relative position duringpolymerase activity can serve as attachment points to a nanopore andtether respectively in an embodiment of the present invention.

In one example, a voltage can be applied across the nanopore 605, e.g.,using measurement circuit 230 and electrodes 231, 232 such as describedfurther above with reference to FIG. 2A, or measurement circuit 240 andelectrodes 241, 242 such as described further above with reference toFIG. 2C. Reporter region 614 or elongated body 613 includes anelectrostatic charge that, responsive to the applied voltage, causeselongated body 613 to extend through constriction 604 such that reporterregion 614 is disposed within or adjacent to constriction 604.Optionally, the applied voltage can cause elongated body 613 to becometaut. As the protein domains of polymerase 650 move, e.g., changeconformation, such movements can impose a force on head region 611,which imposes a force on elongated body 613, which imposes a force onreporter region 614, resulting in translational movement of reporterregion 614 within aperture 603, e.g., movement relative to constriction604. As a result, a conformational change of polymerase 650 can betranslated or transduced into a measurable change in current or fluxthrough aperture 603, which also can be referred to as a blockadecurrent or flux. In one illustrative embodiment, reporter region 614 isconstructed using one or more modified nucleotides. For example, abasicnucleotides typically generate a 70 pA blockade current compared toresidues that include bases, such as dT residues that generate only a 20pA blockade current under conditions that include 10 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer, pH8.0, 300 mM KCl, 1 mM MgCl₂, 1 mM DL-dithiothreitol (DTT), MspA M2mutant pore (D90N, D91N, D93N, D118R, D134R & E139K), 180 mV across a1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) bilayer.

Movements of one or more abasic residues on the order, e.g., of just afew Angstroms, can cause easily detectable changes in current or flux,e.g., of from one to tens of pAs. Because some polymerases move on theorder of nanometers, and a single base in the tether corresponds toabout 0.5 nanometers, it is anticipated that tether movements resultingfrom conformational changes in the polymerase to which the tether isanchored can be generated and readily transduced into currents orfluxes. Because the identity of the nucleotide influences both themagnitude of the conformational change as well as the time spent in theopen state, unique current or flux signatures can be generated thatindividually identify nucleotides as they bind to or reside in theactive site of the polymerase. Additionally, these unique current orflux signatures can individually indicate whether or not a nucleotide iscomplementary or not to a next nucleotide in a polynucleotide beingsequenced. For further details regarding differences in polymeraseconformation and kinetics between match and mismatch nucleotides, seethe following references, the entire contents of each of which areincorporated by reference herein: Freudenthal et al., “New structuralsnapshots provide molecular insights into the mechanism of high fidelityDNA synthesis,” DNA Repair, doi:10.2016/j.dnarep/2015.04.007 (availableonline Apr. 30, 2015); Freudenthal et al., “Watching a DNA polymerase inaction,” Cell Cycle 13: 691-692, doi:10.4161/cc.27789 (2014); andFreudenthal et al., “Observing a DNA polymerase choose right fromwrong,” Cell 154: 157-168, doi:10.1016/j.cell.2013.05.048 (2013).

For example, as illustrated in FIG. 6D, a conformation change ofpolymerase 650 from the open state to the closed state can translate toan “up” movement of reporter region 614 to a first location withinaperture 603, and a conformation change of polymerase 650 from theclosed state to the open state can translate to a “down” movement ofreporter region 614 to a second location within aperture 603, resultingin detectable changes in the blockade current or flux that can becorrelated to individual nucleotides.

For example, a first conformational change of polymerase 650 can occurresponsive to the polymerase binding a first nucleotide, e.g.,nucleotide 661 illustrated in FIG. 6D. The first nucleotide can beindividually identifiable based on a measured (e.g., optically orelectrically measured) magnitude or time, or both, of a first current orflux through the constriction. For example, reporter region 614 can movetowards the polymerase responsive to the first conformational change,causing a change in current or flux through constriction 604.Additionally, a second conformational change of polymerase 650 can occurresponsive to the polymerase binding a second nucleotide. The secondconformational change can differ from the first conformational change,e.g., in magnitude or in time. The second nucleotide can be individuallyidentifiable based on a measured (e.g., optically or electricallymeasured) magnitude or time, or both, of a second current or fluxthrough the constriction. For example, reporter region 614 can movetowards the polymerase responsive to the second conformational change,causing a change in current or flux through constriction 604.

Alternatively, or additionally, a first conformational change ofpolymerase 650 can occur responsive to the polymerase adding a firstnucleotide, e.g., nucleotide 661 illustrated in FIG. 6D, to apolynucleotide, e.g., polynucleotide 660. The first nucleotide can beindividually identifiable based on a measured (e.g., optically orelectrically measured) magnitude or time, or both, of a first current orflux through the constriction. For example, reporter region 614 cantranslationally move towards at least a portion of polymerase 650responsive to the first conformational change, causing a change incurrent or flux through constriction 604. Additionally, a secondconformational change of polymerase 650 can occur responsive to thepolymerase adding a second nucleotide to the polynucleotide. The secondconformational change can differ from the first conformational change,e.g., in magnitude or in time. The second nucleotide can be individuallyidentifiable, and distinguishable from the first nucleotide, based on ameasured (e.g., optically or electrically measured) magnitude or time,or both, of a second current or flux through the constriction. Forexample, reporter region 614 can translationally move towards at least aportion of polymerase 650 responsive to the second conformationalchange, causing a change in current or flux through constriction 604.

As noted above, the magnitude or time duration, or both, of thepolymerase's conformational change(s) can be based on the particularnucleotide that the polymerase receives, binds, and adds to apolynucleotide. Table 1 lists exemplary single molecule kineticparameters that were measured for Klenow fragment processing oftemplates using current changes in a SWNT attached to a singlepolymerase and reported by Olsen et al. In Table 1, τ_(lo) correspondsto the duration of time spent in the polymerase's closed conformation,r_(lo) corresponds to the mean-normalized variance for τ_(lo), τ_(hi)corresponds to the duration of time spent in the polymerase's openconformation, r_(hi) corresponds to the mean-normalized variance forτ_(hi), and the rate corresponds to the rate of processing, e.g., howquickly the polymerase adds the nucleotide to the template. Olson etal., reports that the average magnitude H is a proxy for the extent ofmechanical closure by the enzyme. For the present systems, methods, andcompositions, the value H can be considered to be the extent ofconformational change between two reference points on the polymerase, asmeasured in units of distance.

TABLE 1 (“Template” sequences disclosed as SEQ ID NOS 1-4, respectively,in order of appearance) template nucleotide τ_(lo)(ms) r_(lo)τ_(lzi)(ms) r_(lzi) H (nA) rate (¼) poly(dT)_(a) dATP 0.33 ± 0.08 0.85 ±0.09 71.4 ± 1.4 0.95 ± 0.08 6.94 14.4 ± 2.9 poly(dA)_(a) dTTP 0.42 ±0.09 0.83 ± 0.06 63.7 ± 1.1 0.96 ± 0.06 4.90 16.0 ± 2.9 poly(dG)_(a)dCTP 0.32 ± 0.07 0.78 ± 0.05 30.0 ± 8.6 0.98 ± 0.06 2.53 26.2 ± 4.4poly(dC)_(a) dGTP 0.33 ± 0.05 0.78 ± 0.05 38.0 ± 5.8 1.03 ± 0.07 2.4028.5 ± 3.5 ^(a)Average values ± standard deviation. 7858dx.do|.org/10.1021/ja311603r| J. Am. Chem. Soc. 2013, 135, 7655-7860

Using the compositions, methods, and systems provided herein, a signalthat correlates to the time duration of the open state τ_(hi), themagnitude of conformational change H, and rate of processing togethercan be used to indicate a unique signature for each base. Incorporationrates can also be greatly changed by the selective use modifiednucleotides, such as alpha- or gamma thiol nucleotides. See, forexample, U.S. Patent Publication No. 2011/0312529 to He et al., theentire contents of which are incorporated by reference herein.

In one illustrative embodiment, the template DNA is circularized andpolymerase 650 is a strand-displacing polymerase (such as Phi29). Inthis manner, the template can be sequenced multiple times in a rollingcircle mode such as known in the art. Such an embodiment also caninhibit inadvertently pulling the template DNA into or throughconstriction 604, because only ssDNA can translocate. Any stray ssDNAthat may find its way through constriction 604 (or if a linear templateis used) is expected to transit rapidly and is expected to manifest asnoise in the signal. Alternatively, one can employ a positively chargedreporter region 614 under reverse polarity such that only the reporterregion is drawn into the constriction 604, whereas negatively chargedDNA will be repelled.

In some embodiments, polymerase 650 optionally can be attached, e.g.,anchored, to the mouth of biological pore 605. This can be accomplishedusing cysteine/thiol conjugation chemistry, for example. Such aconjugation can provide that polymerase 650 is anchored in areproducible and stable orientation that can enhance the transfer ofconformational motion of the polymerase 650 to translational motion ofreporter region 614. However, conjugation of the polymerase to the poreneed not be required. For example, the force exerted by the tetherresponsive to the applied voltage can be sufficient to hold thepolymerase in place. In other embodiments, polymerase 650 is notattached to biological pore 605, and tail region 612 can be attached toanother member (such as an oligonucleotide) so as to retain polymerase650 at pore 605.

Additionally, note that a rapid AC current can be used instead of a DCcurrent in order to produce the requisite electric field. This has theadvantage of inhibiting AgCl electrode depletion and lengthening thetime the device can run.

It also should be understood that this approach can be extended to theanalysis of any enzyme or protein that undergoes conformational changes.As such, the present systems, methods, and compositions can beconsidered to provide “nanopore force spectroscopy,” and represent atool that can be used to elucidate enzyme kinetics at the singlemolecule level, and can become an important tool in biochemistryresearch, analytical detection methods and clinical diagnostics.

Exemplary Methods and Compositions for Detecting Action of a PolymeraseUpon a Nucleotide

Techniques other than measurement of a conformational changealternatively can be used to detect events. For example, even if aparticular event can involve a conformational change of a molecule, suchas described above, such an event alternatively, or additionally, can bedetected on another basis. For example, a method can include providing ananopore including a first side, a second side, and an apertureextending through the first and second sides; and providing a permanenttether including a head region, a tail region, and an elongated bodydisposed therebetween. The head region can be anchored to or adjacent tothe first side or second side of the nanopore, and the elongated bodycan include a moiety. The method further can include providing apolymerase disposed adjacent to the first side of the nanopore, andproviding a first nucleotide including a first elongated tag, the firstelongated tag including a moiety. The method further can include actingupon the first nucleotide with the polymerase; and interacting the firstmoiety with the moiety of the tether responsive to the polymerase actingupon the first nucleotide.

In one illustrative example, FIG. 4B illustrates a method for detectingaction of a polymerase upon a nucleotide using a composition including atether anchored to or adjacent to a nanopore, according to someembodiments of the present invention.

Method 410 illustrated in FIG. 4B includes providing a compositionincluding a nanopore, a permanent tether, and a polymerase disposedadjacent to the nanopore (step 411). For example, FIGS. 7A-7Bschematically illustrate a composition including a tether anchored to oradjacent to a nanopore and configured for use in detecting binding of anucleotide by a protein disposed adjacent to the nanopore. In theexemplary embodiment illustrated in FIG. 7A, the composition can includenanopore 700, permanent tether 710, and polymerase 750. Nanopore 700includes first side 701, second side 702, aperture 703, and optionallyalso includes constriction 704. Permanent tether 710 includes headregion 711, tail region 712, and elongated body 713 disposedtherebetween and including reporter region 714 (optionally, one or moreadditional reporter regions can be provided such as described above withreference to FIG. 1C). Polymerase 750 is disposed adjacent to first side701 of nanopore 700. For example, polymerase 750 can be in contact withfirst side 701 of nanopore 700, and optionally can be anchored to oradjacent to the first side of nanopore 700 via any suitable chemicalbond, protein-protein interaction, or any other suitable attachment thatis normally irreversible. Optionally, tail region 712 can be anchored toanother member in a manner analogous to that described with reference toFIGS. 1I and 1M.

In the embodiment illustrated in FIG. 7A, head region 711 of tether 710is attached to, e.g., anchored to, first side 701 of nanopore 700, viaany suitable chemical bond, protein-protein interaction, or any othersuitable attachment that is normally irreversible. Head region 711 canbe attached to any suitable portion of nanopore 700 that places reporterregion 714 within aperture 703 and places elongated tag 713 sufficientlyclose to polymerase 750 so as to interact with nucleotides that can beacted upon by polymerase 750, and optionally also places reporter region714 adjacent to or within constriction 704. For example, nucleotide 730can include an elongated tag 731 including moiety 732 that interactswith tether 710. In an illustrative embodiment, elongated tag 713 oftether 710 can include a moiety 715 with which moiety 732 of tag caninteract. Moiety 715 can be located at any suitable position alongelongated tag 713, e.g., can be located adjacent to head region 711 suchas illustrated in FIG. 7A, or can be adjacent to tail region 712,adjacent to reporter region 714, between head region 711 and reporterregion 714, or between tail region 712 and reporter region 714. Notethat polymerase 750 or nucleotide 730, or both, can be, but need notnecessarily be, considered to be part of the composition, but insteadcan be considered to be in contact with a composition that includesnanopore 700 and permanent tether 710.

Referring again to FIG. 4B, method 410 includes acting upon a nucleotidewith the polymerase (step 412). For example, FIG. 7B schematicallyillustrates binding of nucleotide 730 by polymerase 750, but it shouldbe understood that polymerase 750 can act upon nucleotide 730 in avariety of ways, e.g., by adding nucleotide 730 to a polynucleotide,excising nucleotide 730 from an existing polynucleotide (e.g. viaexonuclease activity or pyrophosphorolysis activity), or samplingnucleotide 730, e.g., transiently interacting with nucleotide 730without binding it. It is anticipated that the dwell time of anucleotide being acted upon by a polymerase can be approximately 1 msecor longer, or 10 msec or longer, or 20 msec or longer, or 50 msec orlonger. The nucleotide can be modified so as to even further extend suchdwell time, e.g., to 50 msec or longer, or 100 msec or longer.

Method 410 illustrated in FIG. 4B also includes interacting a moiety ofthe nucleotide with the tether (step 413). For example, in theembodiment illustrated in FIG. 7B, polymerase 750 acting upon nucleotide730 can bring moiety 732 of nucleotide 730 into sufficiently closeproximity to moiety 715 that the moieties interact with one another,e.g., bond with one another. Such an interaction can be reversible,e.g., can include formation of a hydrogen bond, ionic bond,dipole-dipole bond, London dispersion forces, reversible covalent bond,or any suitable combination thereof

Referring again to FIG. 4B, method 410 also includes moving the reporterregion relative to the constriction responsive to the interaction of thenucleotide's moiety with the tether (step 414). For example, FIG. 7Bschematically illustrates that the interaction between moiety 732 ofnucleotide 730 and moiety 715 of tether 710 can translationally movereporter region 714 toward first side 701, as indicated by the dashedarrow. For example, reporter region 714 can be disposed at a particularlocation within aperture 703 prior to the interaction, e.g., disposedadjacent to or within optional constriction 704 prior to theinteraction, such as illustrated in FIG. 7A, and can be translationallymoved within aperture 703 responsive to interaction between moiety 732and tether 710, e.g., can be translationally moved away fromconstriction 704 toward first side 701. Alternatively, interactionbetween moiety 732 and tether 710 can move head region 711 in such amanner that reporter region 714 moves toward second side 702. It shouldbe appreciated that interaction between moiety 732 and tether 710suitably can cause any type of detectable movement of reporter region714 within aperture 703, e.g., any detectable combination oftranslational, conformational, or rotational movement of reporter region714.

Referring back to FIG. 4B, method 410 further includes detecting themovement of the reporter region within the nanopore aperture (step 415).For example, the composition can be in operable communication with ameasurement circuit such as described above with reference to FIG. 2A orFIG. 2C. The measurement circuit can be configured to detect themovement of the reporter region within the nanopore aperture, e.g.,relative to the constriction. In one illustrative embodiment, nanopore700, tether 710, and polymerase 750 can be immersed in a conductivefluid, e.g., an aqueous salt solution. A measurement circuit configuredanalogously to measurement circuit 230 illustrated in FIG. 2A ormeasurement circuit 240 illustrated in FIG. 2C can be in communicationwith first and second electrodes and can be configured to apply avoltage between those electrodes so as to apply a voltage acrossnanopore 700. The measurement circuit further can be configured to usethe electrodes to measure the magnitude of a current or flux throughaperture 703. Reporter region 714 can have a different electricalproperty than some or all other regions of elongated body 713. Forexample, reporter region 714 can include an electrostatic charge, whilesome or all other regions of elongated body 713 can include a differentelectrostatic charge, or can be uncharged (e.g., can be electricallyneutral). Or, for example, reporter region 714 can be uncharged, whilesome or all other regions of elongated body 713 can include anelectrostatic charge. The magnitude of the current or flux throughaperture 703 can measurably change responsive to translational,rotational, or conformational movement of reporter region 714 within theaperture, e.g., responsive to translational movement of reporter region714 relative to optional constriction 704, and the time period for sucha measurable change in the current or flux can be based on the durationof the reporter region's movement. In one illustrative, nonlimitingexample, elongated body 713 includes a polynucleotide that includes oneor more abasic nucleotides that define reporter region 714.

The action of polymerase 750 upon nucleotide 730 can be individuallyidentifiable based on a measured magnitude or time duration, or both, ofa signal (e.g., optical or electrical signal) generated by such asystem. For example, the action of polymerase 750 upon nucleotide 730can cause reporter region 714 to translationally move to a firstlocation within aperture 703, and the presence of reporter region 714 atthe first location causes the signal to have a first magnitude. As such,the signal having the first magnitude correlates to the action ofpolymerase 750 upon nucleotide 730 having occurred. Note that motions ofreporter region 714 other than translational motion can be detectable,e.g., conformational motion or rotational motion, a combination ofdifferent types of motion.

As illustrated in FIG. 4B, method 410 further can include releasing themoiety of the nucleotide from the tether (step 416). For example, aspolymerase 750 illustrated in FIG. 7B incorporates nucleotide 730 into apolynucleotide, polymerase 750 can cleave elongated tag 731. Suchcleaving can cause dissociation of moieties 732 and 715. Method 410further can include moving the reporter region of the tether responsiveto release of the moiety from the tether (step 417). For example,responsive to release of moiety 732 from moiety 715 illustrated in FIG.7B, reporter region 714 can move translationally toward second side 702,e.g., to a location adjacent to or within constriction 704. Method 410further can include detecting the movement of the reporter region withinthe aperture (step 418). Such detection can be performed analogously asdescribed above with reference to step 415. Note that motions ofreporter region 714 other than translational motion can be detectable,e.g., conformational motion or rotational motion.

Sequencing by Synthesis Using Exemplary Methods and Compositions Basedon Detecting Action of Polymerases Upon Nucleotides

It should be appreciated that method 410 illustrated in FIG. 4B suitablycan be used to detect the action of one type of molecule upon any othersuitable type of molecule having a moiety attached thereto.

In one nonlimiting, illustrative embodiment described below withreference to FIGS. 8A-14, method 410 can be used to detect apolymerase's action upon a nucleotide. Detection of such action can beused to sequence a first polynucleotide by synthesizing a secondpolynucleotide that is complementary to the first nucleotide, e.g.,using “sequencing by synthesis” (SBS).

Under one aspect, a composition can include a nanopore including a firstside, a second side, and an aperture extending through the first andsecond sides; and a permanent tether including a head region, a tailregion, and an elongated body disposed therebetween. The head region canbe anchored to or adjacent to the first side or second side of thenanopore, and the elongated body can include a moiety. A polymerase canbe disposed adjacent to the first side of the nanopore. The compositionalso includes a first nucleotide including a first elongated tag. Thefirst elongated tag includes a first moiety that interacts with themoiety of the tether responsive to the polymerase acting upon the firstnucleotide.

In one illustrative example, FIG. 8A schematically illustrates anexemplary composition including a tether anchored to a nanopore andconfigured for use in detecting action of a polymerase upon anucleotide. The nanopore includes biological pore 805, which can bedisposed in a barrier (not specifically illustrated), e.g., a membraneof biological origin such as a lipid bilayer, or a solid state membrane.Biological pore 805 includes aperture 803 and constriction 804, althoughit should be understood that biological pore 805 suitably can include noconstriction, or multiple constrictions. The permanent tether includeshead region 811, elongated body 813, and reporter region 814. Polymerase850 is disposed adjacent to, and in contact with, biological pore 805,and optionally can be anchored to biological pore 805 via a physical orchemical linkage (e.g., using click chemistry or a cysteine-maleimidebond). Polymerase 850 is configured to receive a template polynucleotide860, e.g., circular or linear ssDNA to be sequenced, to synthesize apolynucleotide having a complementary sequence to that of the ssDNA bysequentially acting upon nucleotides, e.g., binding nucleotides, addingthe nucleotides to a polynucleotide in accordance with the sequence ofthe ssDNA, excising the nucleotides from an existing polynucleotide, orby sampling the nucleotides, e.g., transiently interacting with thenucleotides without binding them. Head region 811 can be anchored to anysuitable portion of nanopore 800 that places reporter region 814 withinaperture 803, e.g., adjacent to or within constriction 804 and placeselongated body 813 sufficiently close to polymerase 850 so as tointeract with nucleotides that can be acted upon by polymerase 850. Forexample, nucleotide 830 can include an elongated tag 831 includingmoiety 832 that interacts with moiety 815 of the tether. The templateDNA to be sequenced and the primer for the complementary polynucleotideto be sequenced are represented in FIG. 8A by the black lines (thebroken line indicating a relatively long distance).

In one example, a voltage can be applied across the nanopore 805, e.g.,using measurement circuit 230 and electrodes 231, 232 such as describedfurther above with reference to FIG. 2A, or measurement circuit 240 andelectrodes 241, 242 such as described further above with reference toFIG. 2C. Reporter region 814 or elongated body 813 includes anelectrostatic charge that, responsive to the applied voltage, causeselongated body 813 to extend through aperture 803, optionally such thatreporter region 814 is disposed within or adjacent to constriction 804.Optionally, the applied voltage can cause elongated body 813 to becometaut. Responsive to polymerase 805 acting upon nucleotide 830, moiety832 of nucleotide 830 can interact with, e.g., reversibly bond to,moiety 815 of tether 832. Such interaction can impose a force onreporter region 814 resulting in movement of reporter region 814 withinaperture 803, e.g., translational movement. As a result, the action ofpolymerase 850 upon nucleotide 830 can be translated or transduced intoa measurable change in current or flux through constriction 804, whichalso can be referred to as a blockade current or flux. Additionally, theforce exerted on tether by the applied voltage can be expected to pullon the pore rather than the polymerase, and thus is not expected tosignificantly disrupt polymerase activity.

In one illustrative embodiment, moiety 815 includes a firstoligonucleotide, and moiety 832 includes a second oligonucleotide thatis complementary to the first oligonucleotide, e.g., that hybridizes tothe first oligonucleotide responsive to the action of polymerase 850upon nucleotide 830. The hybridization of the second oligonucleotide tothe first oligonucleotide can cause a change in the length of theelongated body 813 of tether 810, which in turn can move reporter region814 to a predetermined location. The action of polymerase 850 uponnucleotide 830 can be individually detected based on a measured (e.g.,optically or electrically measured) magnitude or time duration, or both,of a current or flux through aperture 803. In one illustrativeembodiment, moiety 815 includes a first oligonucleotide, and moiety 832includes a second oligonucleotide that is complementary to the firstoligonucleotide, e.g., that hybridizes to the first oligonucleotide. Thehybridization of the second oligonucleotide to the first oligonucleotidecan shorten the elongated body 813 of tether 810 by a predeterminedamount, which in turn can move reporter region 814 to a predeterminedlocation within aperture 803. The binding of the nucleotide can beindividually detected based on a measured (e.g., optically orelectrically measured) magnitude or time duration, or both, of a currentor flux through aperture 803.

For example, FIG. 8B schematically illustrates an exemplary nucleotideincluding an elongated tag including a moiety that interacts with thetether of FIG. 8A during use in detecting action of a polymerase uponthe nucleotide. As illustrated in FIG. 8B, elongated tag 831 ofnucleotide 830, e.g., T, can include an oligonucleotide moiety 832attached to the gamma phosphate of the nucleotide 830, e.g., via a deltaphosphate linkage. Oligonucleotide moiety 832 can include any suitablesequence of nucleotides selected to hybridize to a correspondingsequence of nucleotides within moiety 815 of the tether. For example,oligonucleotide moiety 832 illustrated in FIG. 8B can include theexemplary sequence 5′ GCAT 3′, and moiety 815 can include thecomplementary sequence 5′ ATGC 3′. Referring again to FIG. 8A, theaction of polymerase 805 upon nucleotide 830 can maintain moiety 832 inrelatively close proximity to moiety 815 of the tether, resulting in atransient increase in the local concentration of oligonucleotide moiety832 that can induce hybridization between moieties 832 and 815preferentially to moieties that are attached to nucleotides notpresently being acted upon by polymerase 805. The resultinghybridization causes movement of the tether, e.g., conformationalmovement resulting in a shortening of elongated body 813 that can movereporter region 814 relative to constriction 804. Polymerase 850 cancleave elongated tag 831 upon incorporating nucleotide 830 into apolynucleotide, responsive to which moiety 832 can dissociate frommoiety 815.

The conformational change in the tether can be induced by the creationof double stranded DNA (dsDNA) from ssDNA that respectively is includedwithin moieties 815 and 832. For example, ssDNA is longer than dsDNA byabout 1.5 Angstroms per nucleotide, which is within the resolutionlimits of the present systems, e.g., system 220 illustrated in FIG. 2Aor system 250 illustrated in FIG. 2C. Each nucleotide can include acorresponding oligonucleotide moiety 832 that is selected to create adifferent length of dsDNA upon hybridization of moiety 832 with moiety815, thus shortening the tether by a distance that corresponds to thenucleotide being acted upon by polymerase 850. In some embodiments, theformula for the amount of shortening of a fully taut tether, such as atether extended across the pore responsive to an applied voltage, can beexpressed as:

D _(s) =N*(L _(ss) −L _(ds))  (1)

where N is the number of bases that are hybridized, D_(s) is thedistance by which the tether shortens, L_(ss) is the length betweennucleotides in ssDNA (approximately 5 Angstroms), and L_(ds) is thelength between nucleotides in dsDNA (approximately 3.3 Angstroms).

FIGS. 9A-9B schematically illustrate a conformational change, e.g.,shortening, of an exemplary tether responsive to hybridization with amoiety of an elongated tag of an exemplary nucleotide during use indetecting action of a polymerase upon the nucleotide. Head region 911 ofthe tether is anchored to nanopore 900 optionally including constriction904, and elongated body 913 extends through aperture 903, e.g., suchthat reporter region 914 is disposed within, or adjacent to,constriction 914. In the embodiment illustrated in FIGS. 9A-9B,elongated body 913 includes a polynucleotide such as ssDNA, thenucleotides of which are represented by horizontal bars 916. Reporterregion 914 includes one or more abasic sites of the polynucleotide,e.g., ssDNA. Moiety 915 includes a sequence of nucleotides that isselected so as to hybridize with a corresponding moiety, e.g., acomplementary sequence of nucleotides, of an elongated tag of anucleotide being acted upon by polymerase 900 (nucleotide being actedupon, and elongated tag thereof, not specifically illustrated in FIGS.9A-9B).

As illustrated in FIG. 9A, prior to binding of moiety 915 to thecorresponding moiety of the nucleotide being acted upon, nucleotides 916are spaced apart from one another by approximately 5 Angstroms. Asillustrated in FIG. 9B, responsive to moiety 915 interacting with, e.g.,hybridizing to, the corresponding moiety of the nucleotide being actedupon, e.g., responsive to the moieties forming a double stranded DNAduplex, the spacing between nucleotides 916 within moiety 915 decreasesto about 3.3 Angstroms. The short, black vertical line in FIG. 9Bindicates a 5-base hybridization event, which shortens the tether andmoves reporter region 914 to a new location. Duplexes of 6, 7 or 8 basescan be even shorter than represented in FIG. 9B, as discussed furtherbelow.

Each different type of nucleotide can include a corresponding elongatedtag that is attached to its gamma phosphate in a manner analogous tothat illustrated in FIG. 8B, or otherwise suitably attached. Forexample, FIGS. 10A-10B schematically illustrate exemplary nucleotidesincluding elongated tags including respective moieties that interactwith an exemplary tether during use in detecting binding of thenucleotide by a polymerase disposed adjacent to a nanopore. As shown inFIG. 10A, the A, T, C, and G nucleotides can include respectivelyelongated tags that include different moieties than one another, e.g.,as respectively represented by the triangle, diamond, square, andcircle. The particular moieties can be suitably selected so as tointeract with, e.g., hybridize to, a corresponding moiety of thepermanent tether, and to induce different respective conformationalchanges to the tether. FIG. 10B illustrates nonlimiting examples ofmoieties that can be included in the elongated tags illustrated in FIG.10A. Each such moiety can interact with, e.g., hybridize with, adifferent, respective portion of a corresponding moiety of the permanenttether, so as to induce a different, respective conformational change ofthe tether.

For example, FIG. 10C schematically illustrates an exemplary tether thatincludes head region 1011, tail region 1012, and elongated body 1013that includes reporter region 1014 and moiety 1015. Head region 1011 caninclude a chemical linker such as a 3′ maleimide (“Mal”) group forconjugation to a cysteine (Cys) residue on the pore. Tail region 1012can include a 5′ phosphate group (“phos”) that is charged and thusassists with feeding the tether through the aperture of the poreresponsive to an applied voltage. Elongated body 1013 can include apolymer, e.g., a polynucleotide such as illustrated in FIG. 10C, or anyother suitably polymer, such as a biological polymer or a syntheticpolymer. Reporter region 1014 includes one or more abasic nucleotidesdenoted as “X”, and in one exemplary embodiment can be located about14-15 bases from the maleimide, which can be about the distance H2 fromthe pore mouth to the pore constriction for certain nanopore types.Moiety 1015 can include a sequence of nucleotides, e.g., GGGTATAT, withwhich each of the moieties attached to the A, T, C, and G nucleotides tobe acted upon can interact, e.g., hybridize, differently than oneanother.

Note that the moieties illustrated in FIGS. 10A-10C are intended to bepurely exemplary, and not limiting of the invention. However, themoieties attached to the nucleotides to be acted upon can be selected soas satisfy one or more of the following parameters, and optionally allof the following parameters:

-   -   1. Moieties attached to different types of nucleotides than one        another can interact with the moiety of the tether in a manner        that is distinguishable from one another, e.g., via measurement        of current or flux through the pore constriction.    -   2. The stability of a duplex between the moiety of the        nucleotide and the corresponding moiety of the tether is        sufficiently low that such moieties attached to “free”        nucleotides (nucleotides that are not being acted upon by the        polymerase and thus transiently interact with the tether)        interact only briefly with the moiety of the tether, e.g., for        less than 1 msec. For example, the stability of the duplex        between the moiety of the nucleotide and the moiety of the        tether can be expressed as the Tm, or melting temperature, of        the duplex. The system operational temperature is expected to be        about 20° C., or room temperature. Moieties that are about 5-8        nucleotides long are expected have Tm<12° C., which can provide        sufficiently low stability at room temperature that moieties        attached to “free” nucleotides will interact only briefly with        the moiety of the tether.    -   3. The stability of a duplex between the moiety of the        nucleotide and the corresponding moiety of the tether is        sufficiently high that when the nucleotide is acted upon and        held in place by the polymerase for the several milliseconds (1        to 30 msec, for example) during incorporation, such action        increases the effective concentration of the moiety of the        nucleotide relative to the moiety of the tether, which drives        the reaction between the moieties forward and increases        stability such that the effective Tm of the duplex is greater        than 20° C. (or the anticipated operational temperature of the        system), e.g., is greater than 30° C., or greater than 40° C.,        or greater than 50° C.    -   4. The length of the elongated tag of the nucleotide being acted        upon, e.g., the length between the moiety and the gamma        phosphate, can be sufficiently long that when the moiety is        stably hybridized to the corresponding moiety of the tether,        there is substantially no force on the nucleotide. If this        length is too short, the tether can impose a force on the        elongated tag of the nucleotide, which is expected to result in        reduced polymerase efficiency.

For further information about hybridizing oligonucleotides to oneanother, see U.S. Pat. No. 8,652,779 to Turner et al., the entirecontents of which are incorporated by reference herein. According toTurner et al., at such a size scale, an oligonucleotide should sampleits configuration space about 100-fold faster than a polymerase canincorporate a nucleotide. Applying such a principle to the presentcompositions, it is believed to be likely that the moiety of thenucleotide being acted upon will readily “find,” and interact with, thecorresponding moiety of the tether, and also will dissociate from thetether after the moiety is cleaved from the nucleotide being acted upon.

Note that in the exemplary moieties illustrated in FIG. 10C, each moietyattached to a nucleotide being acted upon has only a single matchinghybridization with the corresponding tether moiety 1015 that includesbetween 5 to 8 bases. While other hybridization options exist that donot cause complete hybridization, such options can be anticipated to besignificantly less stable than the full-length options.

FIGS. 11A-11D illustrate exemplary calculations of interactions betweena tether and moieties. Hybridization options are shown for each moietywith their predicted free energies, based upon the assumption of twofreely diffusing oligonucleotides in the presence of 50 mM NaCl and 2 mMMg²⁺, a divalent ion that is known to increase Tm and can be used atthis concentration for polymerase activity. The “tether sequence”illustrated above FIGS. 11A-11D corresponds to the sequence of thetether, and the “tag sequences” illustrated in FIGS. 11A-11D correspondto exemplary sequences of moieties that respectively can be attached toa nucleotide. The moiety (tag sequence) illustrated in FIG. 11A is fivebase pairs long, while the moiety (tag sequence) illustrated in FIG. 11Bis six base pairs long and is similar to the moiety illustrated in FIG.11A but includes one additional base, A, on the 3′ end. The moiety (tagsequence) in FIG. 11C is seven base pairs long and is similar to themoiety illustrated in FIG. 11B but includes one additional base, T, onthe 3′ end. The moiety (tag sequence) in FIG. 11D is eight base pairslong and is similar to the moiety illustrated in FIG. 11C but includesone additional base, A, on the 3′ end.

The calculated difference in free energy (ΔG) is respectivelyillustrated for the best match (solid box) and second best match (dashedbox) hybridizations between the primary sequence and the differentsecondary sequences, based on the assumption that the primary sequenceand the respective secondary sequence are freely diffusing. Morespecifically, in FIG. 11A it may be seen that the calculated ΔG for theenergetically most favorable exemplary hybridization illustrated in box1A was −9.57 kcal/mole, corresponding to hybridization of all 5 basepairs of the respective tag sequence with the tether sequence, while thecalculated ΔG for the energetically second most favorable exemplaryhybridization illustrated in box 1B was −3.07 kcal/mole, correspondingto hybridization of only 2 base pairs of that tag sequence with thetether sequence. In FIG. 11B it may be seen that the calculated ΔG forthe energetically most favorable exemplary hybridization illustrated inbox 2A was −10.53 kcal/mole, corresponding to hybridization of all 6base pairs of the respective tag sequence with the tether sequence,while the calculated ΔG for the energetically second most favorableexemplary hybridization illustrated in box 2B was −3.2 kcal/mole,corresponding to hybridization of only 3 base pairs of that tag sequencewith the tether sequence. In FIG. 11C it may be seen that the calculatedΔG for the energetically most favorable exemplary hybridizationillustrated in box 3A was −12 kcal/mole, corresponding to hybridizationof all 7 base pairs of the respective tag sequence with the tethersequence, while the calculated ΔG for the energetically second mostfavorable exemplary hybridization illustrated in box 3B was −3.2kcal/mole, corresponding to hybridization of only 3 base pairs of thattag sequence with the tether sequence. In FIG. 11D it may be seen thatthe calculated ΔG for the energetically most favorable exemplaryhybridization illustrated in box 4A was −12.96 kcal/mole, correspondingto hybridization of all 8 base pairs of the respective tag sequence withthe tether sequence, while the calculated ΔG for the energeticallysecond most favorable exemplary hybridization illustrated in box 4B was−3.2 kcal/mole, corresponding to hybridization of only 3 base pairs ofthat tag sequence with the tether sequence. Accordingly, it can beunderstood from FIGS. 11A-11D that the calculated ΔG is significantlylower for full hybridization of each of the secondary sequences to theprimary sequence than for a partial hybridization of those tag sequencesto the tether sequence. It further can be understood from FIGS. 11A-11Dthat the predicted melting temperatures (Tms) are all less thanapproximately 11.4° C., including for the moiety with 8 bases. At 150 mMsalt (a concentration that suitably can be used in systems configured tomeasure current or flux through a pore aperture) and 2 mM Mg2+, themoiety with 8 bases has a predicted Tm of approximately 15° C. Thus, itcan be expected that freely diffusing nucleotides that encounter thetether will have no meaningful stability at room temperature.Additionally, the highest Tm can be lowered even further, e.g., byapproximately 5° C., by using, for example, formamide.

Additionally, it can be expected that the melting temperatures ofduplexes between moieties on nucleotides being acted upon, and moietieson the tether, to be significantly more stable than an otherwiseidentical pair of freely diffusing oligonucleotides because the tetherand the incorporating nucleotide are held in relatively fixed positionrelative to another, causing an effective increase in the localconcentrations of the moieties. The resulting synergistic binding canoccur because the nucleotide is held simultaneously to some extent byboth the polymerase and the hybridization interaction. Such synergisticbinding can significantly increase the effective Tm of a shortoligonucleotide duplex. FIG. 12A illustrates a model that can be used tocalculate interactions between a tether and moieties. Such a model isbased on a molecular beacon, in which two short oligonucleotides ofabout 5 or 6 bases each are stably hybridized with one another in astem-loop structure. The loop serves to hold the two stem pieces (theoligonucleotides) in close proximity to one another. Tms>50° C. or >60°C. can be readily achieved with stems of about 5-6 nucleotides and loopson the order of 10 or more nucleotides. Because the oligonucleotides areheld in relatively close proximity to one another by the loop (“probesequence”), their effective concentration is increased and cansignificantly increase the Tm of hybridization between those nucleotidesas compared to freely diffusing nucleotides.

FIG. 12B illustrates exemplary calculations of interactions between atether and moieties that can be attached to a nucleotide being actedupon by a polymerase, based on the model of FIG. 12A, and FIG. 12Cillustrates a stable structure calculated based on the model of FIG.12A. More specifically, using the example of the sequence encoding themoiety of FIG. 10B for nucleotide “A”, the mFold program hosted by theRNA Institute (College of Arts and Sciences, University of Albany, StateUniversity of New York at Albany) was used to determine the Tm of thissequence hybridized to its reverse complement in the tether assuming aloop that is 10 nucleotides long. It is anticipated that such a lengthof the loop is a reasonable approximation for the length of otherportions of the elongated tag of the nucleotide being acted upon by thepolymerase. As shown in FIG. 12B, the predicted Tm in the presence of 2mM MgCl₂ and 50 mM NaCl is approximately 49° C., indicating that evenrelatively short 5-mer moieties such as illustrated in FIG. 12C can haverelatively high Tms if the effective concentration of the moietiesrelative to one another is sufficiently high. The exemplary moieties forthe other bases (C, G & T) illustrated in FIG. 10B are even longer andrange from 6-8 nucleotides, and thus can be expected to have somewhathigher Tms. For example, “G” with an 8-nucleotide moiety is predicted tohave a Tm of 59° C. in 2 mM Mg² and 50 mM NaCl (data not shown) based onthe model illustrated in FIG. 12A. In comparison, FIG. 10C illustratesthe respective Tm for freely diffusing forms each of the illustratedsequences as predicted by the mFold program in the presence of 1 mM Mg²⁺and 150 mM NaCl. Note that the mFold program does not report values<10°C. All Tms for freely diffusing forms are well below the expectedoperating temperature of the system at room temperature (approximately20° C.), while Tms calculated using the model of FIG. 12A are well abovethe expected operated temperature of the system.

As noted above, oligonucleotide moieties of different lengths can beused to change the conformation of the tether, e.g., to change thelength of the tether, e.g., shorten the tether, by differing amounts.Applying equation (1) above to moieties ranging from 5 to 8 nucleotidesyields the results in Table 2. The moiety having 5 nucleotides isanticipated to shorten the tether by approximately 8.5 Angstroms. Themoiety having 6 nucleotides is anticipated to shorten the tether byapproximately 10.2 Angstroms. The moiety having 7 nucleotides isanticipated to shorten the tether by approximately 11.9 Angstroms. Themoiety having 8 nucleotides is expected to shorten the tether byapproximately 13.6 Angstroms. Accordingly, moieties shorten the tetherby respective amounts that are “spaced” from one another byapproximately 1.7 Angstroms.

TABLE 2 Differential shortening from labels of 5-8 bases. Label LengthDs (Ang) Difference from shortest label (Ang) 5 8.5 N/A 6 10.2 1.7 711.9 1.7 8 13.6 1.7

Such conformational movements of the tether responsive to interactionswith moieties attached to nucleotides being acted upon by a polymerasecan provide signals that facilitate identification of differentnucleotides as the polymerase adds such nucleotides to a polynucleotide,e.g., during sequencing by synthesis. FIGS. 13A-13E schematicallyillustrate movement of an exemplary tether within the aperture of a pore(e.g., nanopore) responsive to interactions with exemplary moieties ofrespective nucleotides, such as the moieties illustrated in FIGS.10A-10C. Note that in FIGS. 13A-13E, the polymerase is not specificallyillustrated, but can be located adjacent to the first side of the porein a manner analogous to that described above with reference to FIG. 8A.Additionally, in FIGS. 13B-13E, the nucleotide being acted upon by thepolymerase is not specifically illustrated, but can be located withinthe polymerase in a manner analogous to that described above withreference to FIG. 8A. The dotted line in FIGS. 13B-13E is intended torepresent portions of the elongated tag of the nucleotide being actedupon that connect the moiety to the nucleotide. Additionally, in FIGS.13A-13E, measurement circuitry configured to measure (e.g., optically orelectrically measure) movements of the reporter region within theaperture of the pore is not specifically illustrated, but can beconfigured in a manner analogous to that described above with referenceto FIG. 2A or FIG. 2C. In one illustrative embodiment, the measurementcircuitry is configured to apply a voltage across the pore and tomeasure current or flux through the aperture of the pore.

FIG. 13A illustrates the pore and tether in the absence of an event,which can be referred to as their equilibrium state. The reporterregion, e.g., one or more abasic residues, denoted by “X,” resideswithin the aperture of the pore, e.g., within the constriction of thepore. FIG. 14 is a plot of an exemplary signal that can be generatedduring the interactions illustrated in FIG. 13A-13E. As illustrated inFIG. 14, the signal (e.g., optically or electrically measured current orflux) between times t₀ and t₁ can have a first value (A) correspondingto the location of the reporter region illustrated in FIG. 13A. FIG. 13Billustrates an interaction between an exemplary oligonucleotide moietyattached to a “dA” nucleotide, which is being acted upon by thepolymerase, with a corresponding oligonucleotide moiety on the tether attime t₁. Based on equation (1), it is anticipated that, responsive tothe interaction, the tether can change configuration, e.g., shorten, insuch a manner that causes reporter region “X” to move within theaperture of the pore by approximately 8.5 Angstroms toward the firstside of the pore, e.g., in the direction of the polymerase. Asillustrated in FIG. 14, the signal (e.g., optically or electricallymeasured current or flux) between times t₁ and t₂ can have a secondvalue (B) corresponding to the location of the reporter regionillustrated in FIG. 13B. At approximately time t₂, the polymerasecleaves the elongated tag from the dA nucleotide upon which thepolymerase is acting, responsive to which the moiety (formerly) of thedA nucleotide dissociates from the tether, responsive to which thereporter region returns to the state illustrated in FIG. 13A and thesignal returns to value (A) until the polymerase acts on anothernucleotide. Note that signal changes, e.g., current or flux changes, inFIG. 14 are illustrated as step functions for simplicity, but it shouldbe appreciated that the current or flux changes can have more complexshapes based on the particular manner in which the nucleotide is actedupon by the polymerase, and thus the particular manner in which thereporter region moves within the aperture of the pore. Additionally,noise can be present in signals such as illustrated in FIG. 14 and canmanifest as transient spikes (not shown).

FIG. 13C illustrates an interaction between an exemplary oligonucleotidemoiety attached to a “dT” nucleotide, which is being acted upon by thepolymerase, with the corresponding oligonucleotide moiety on the tetherat time t₃. Based on equation (1), it is anticipated that, responsive tothe interaction, the tether will shorten in such a manner that causesreporter region “X” to move within the aperture of the pore byapproximately 10.2 Angstroms toward the first side of the pore, e.g., inthe direction of the polymerase. As illustrated in FIG. 14, the signal(e.g., optically or electrically measured current or flux) between timest₃ and t₄ can have a third value (C) corresponding to the location ofthe reporter region illustrated in FIG. 13C. At approximately time t₄,the polymerase cleaves the elongated tag from the dT nucleotide uponwhich the polymerase is acting, responsive to which the moiety(formerly) of the dT nucleotide dissociates from the tether, responsiveto which the reporter region returns to the state illustrated in FIG.13A and the signal returns to value (A).

FIG. 13D illustrates an interaction between an exemplary oligonucleotidemoiety attached to a “dC” nucleotide, which is being acted upon by thepolymerase, with the corresponding oligonucleotide moiety on the tetherat time t₅. Based on equation (1), it is anticipated that, responsive tothe interaction, the tether will shorten in such a manner that causesreporter region “X” to move within the aperture of the pore byapproximately 11.9 Angstroms toward the first side of the pore, e.g., inthe direction of the polymerase. As illustrated in FIG. 14, the signal(e.g., optically or electrically measured current or flux) between timest₅ and t₆ can have a fourth value (D) corresponding to the location ofthe reporter region illustrated in FIG. 13D. At approximately time t₆,the polymerase cleaves the elongated tag from the dC nucleotide uponwhich the polymerase is acting, responsive to which the moiety(formerly) of the dC nucleotide dissociates from the tether, responsiveto which the reporter region returns to the state illustrated in FIG.13A and the signal returns to value (A).

FIG. 13E illustrates an interaction between an exemplary oligonucleotidemoiety attached to a “dG” nucleotide, which is being acted upon by thepolymerase, with the corresponding oligonucleotide moiety on the tetherat time t₇. Based on equation (1), it is anticipated that, responsive tothe interaction, the tether will shorten in such a manner that causesreporter region “X” to move within the aperture of the pore byapproximately 13.6 Angstroms toward the first side of the pore, e.g., inthe direction of the polymerase. As illustrated in FIG. 14, the signal(e.g., optically or electrically measured current or flux) beginning attime t₇ can have a fifth value (E) corresponding to the location of thereporter region illustrated in FIG. 13E. At approximately time t₈, thepolymerase cleaves the elongated tag from the dG nucleotide upon whichthe polymerase is acting, responsive to which the moiety (formerly) ofthe dC nucleotide dissociates from the tether, responsive to which thereporter region returns to the state illustrated in FIG. 13A and thesignal returns to value (A) (not specifically illustrated in FIG. 14).

Table 3 lists exemplary moieties that can be included in the elongatedtag of each nucleotide and that interacts with a corresponding moietyalong the elongated body of the tether, e.g., the moieties illustratedin FIGS. 13A-13E. Table 3 also lists the number of nucleotides in themoiety that hybridize (“hybed”) with the corresponding moiety of thetether. Table 3 also lists the magnitude of the resulting conformationalchange to the tether, e.g., the amount by which the tether isanticipated to be shortened, in Angstroms. Table 3 also lists theexpected differential length of the tether with respect to the exemplarymoiety of “dA”. Assuming that single stranded DNA has 5 Angstrom spacingbetween nucleotides, the equivalent shortening length measured in termsof single stranded DNA also is listed in Table 3, as well as thedifferential distance between nucleotides with respect to “dA”, measuredin nucleotides (“Delta Bases”).

TABLE 3 Label Seq Num. Hybed Ang. Delta # ssDNA Bases Delta Base (5′-3′)Bases Shortening Ang. Equiv Bases A CCCAT 5 8.5 N/A 1.7 N/A T CCCATA 610.2 1.7 2.04 0.34 C CCCATAT 7 11.9 1.7 2.38 0.34 G CCCATATA 8 13.6 1.72.72 0.34

Additionally, note that because the moieties of the nucleotides beingacted upon can be of different lengths, e.g., ranging from 5 to 8nucleotides, their Tms can differ somewhat from each other. The Tm anddelta G of a given moiety is expected to factor into the rate at whichthe moiety of the nucleotide dissociates from the moiety of the tether(also referred to as the off rate). Such a rate, or time duration, canpotentially be used as another characteristic to ascertain the correctidentity of each nucleotide. For example, a signal having a time periodcorresponding to the difference between t₁ and t₂ illustrated in FIG. 14can be correlated to the polymerase acting upon dA in a manner analogousto that illustrated in FIG. 13B, a signal having a time periodcorresponding to the difference between t₃ and t₄ can be correlated tothe polymerase acting upon dT in a manner analogous to that illustratedin FIG. 13C, a signal having a time period corresponding to thedifference between t₅ and t₆ can be correlated to the polymerase actingupon dC in a manner analogous to that illustrated in FIG. 13D, or asignal having a time period corresponding to the difference between t₇and t₈ can be correlated to the polymerase acting upon dG in a manneranalogous to that illustrated in FIG. 13E.

Note that the duplex formed between the moiety of the elongated tag andthe moiety of the tether can be in thermodynamic equilibrium. It shouldbe appreciated that a duplex in thermodynamic equilibrium can have onand off rates that are based upon the length and character of thenucleic acid sequence, and that the duplex may dissociate fromtime-to-time. It can be useful for the mean time spent in the duplexstate (the inverse of the off rate) to be shorter than the averagelifetime of the polymerase-nucleotide complex during the incorporationevent, so that after incorporation and tag release from the nucleotide,the tag will diffuse away and not block incoming nucleotide tags. Theeffective on rate can be sufficiently high to result in relatively fastre-binding as compared with the lifetime of the polymerase-nucleotidecomplex, so that incorporation events are detected and so that theduplex reforms after any dissociation events occurring during nucleotideincorporation. The on rate will be pseudo-first order in theconcentration of the elongated tag of the nucleotide, which can beconsidered to make such an arrangement a stochastic sensor of theconcentration of the elongated tag. Note that freely diffusing elongatedtags can have a relatively low concentration (e.g., from 10 nM to 100nM, or from 100 nM to 250 nM, or from 250 nM to 500 nM, or from 500 nMto 1 uM), whereas the elongated tag of the nucleotide being acted uponwill effectively have a relatively high concentration because it isbound to the nucleotide which is held in place by the polymerase duringincorporation, and thus is not free to diffuse away.

Accordingly, it should be appreciated that the reporter region of one ofthe present tethers is movable (e.g., translationally movable) within anaperture by different amounts, or for different amounts of time, orboth, responsive to the polymerase acting upon different nucleotides.Such nucleotides can be individually identifiable based on a measured(e.g., optically or electrically measured) magnitude or time duration,or both, of a signal, e.g., of a current or flux through the aperture.For example, first and second nucleotides can be attached to moietiesthat interact with the tether differently than one another. For example,the tether can include a first oligonucleotide, and the moiety attachedto the first nucleotide can include a second oligonucleotide thathybridizes to the first oligonucleotide so as to move the reporterregion toward the first side responsive the polymerase acting upon thefirst oligonucleotide, e.g., to shorten the tether by a first amount.The moiety attached to the second nucleotide can include a thirdoligonucleotide that hybridizes to the first oligonucleotide so as tomove the reporter region toward the first side responsive the polymeraseacting upon the second oligonucleotide, e.g., to shorten the tether by asecond amount. The first and second nucleotides can be distinguishablefrom one another, e.g., the first nucleotide can be individuallyidentifiable based on a measured (e.g., optically or electricallymeasured) magnitude or time duration, or both, of a first signal, e.g.,of a first current or flux through the constriction, and the secondnucleotide can be individually identifiable based on a measured (e.g.,optically or electrically measured) magnitude or time duration, or both,of a second signal, e.g., of a second current or flux through theconstriction.

In the exemplary embodiments described above with reference to FIGS.8A-14, the elongated body of the tether and the elongated tag of thenucleotide being acted upon by the polymerase respectively can include,or can even consist solely of, single-stranded DNA (ssDNA). However, itshould be appreciated that other types of molecules suitably can beused. For example, any tether suitably can be used that includes anelongated body having one or more of the following features, andoptionally includes all of the following features:

-   -   1. The elongated body can include a region that interacts with        moieties respectively attached to different types of nucleotides        in a manner that the moieties are distinguishable from one        another, e.g., via measurement of current or flux through the        pore constriction.    -   2. The elongated body can include a charged region that causes        it to be pulled through the constriction of the pore responsive        to an applied voltage. The elongated body can be held taut in        such a configuration. This charged region can be located        adjacent to the pore constriction to result in a net force.    -   3. The elongated body includes a reporter region that when moved        through the pore aperture, e.g., through the pore constriction,        yields a clearly distinguishable signal. The reporter region and        the charged region can be the same as one another; that is, a        single region can be both a reporter region and a charged        region.

An exemplary material that can be included in the elongated body of thetether, or the elongated tag of the nucleotide being acted upon, orboth, is a polymer. Polymers include biological polymers and syntheticpolymers. Exemplary biological polymers that are suitable for use in theelongated body of the tether, or the elongated tag of the nucleotidebeing acted upon, or both, include polynucleotides, polypeptides,polysaccharides, polynucleotide analogs, and polypeptide analogs.Exemplary polynucleotides and polynucleotide analogs suitable for use inthe elongated body of the tether, or the elongated tag of the nucleotidebeing acted upon, or both, include DNA, enantiomeric DNA, RNA, PNA(peptide-nucleic acid), morpholinos, and LNA (locked nucleic acid).Exemplary synthetic polypeptides can include charged amino acids as wellas hydrophilic and neutral residues. In some embodiments, the tether isnot a nucleic acid or does not include nucleotides. For example, atether can exclude naturally occurring nucleotides, non-naturallyoccurring nucleotide analogs, or both. One or more of the nucleotidesset forth herein or otherwise known in the art can be excluded from atether.

Other exemplary polymers that can be suitable for use in the elongatedbody of the tether, or the elongated tag of the nucleotide being actedupon, or both, include synthetic polymers such as PEG (polyethyleneglycol), PPG (polypropylene glycol), PVA (polyvinyl alcohol), PE(polyethylene), LDPE (low density polyethylene), HDPE (high densitypolyethylene), polypropylene, PVC (polyvinyl chloride), PS(polystyrene), NYLON (aliphatic polyamides), TEFLON®(tetrafluoroethylene), thermoplastic polyurethanes, polyaldehydes,polyolefins, poly(ethylene oxides), poly(w-alkenoic acid esters),poly(alkyl methacrylates), and other polymeric chemical and biologicallinkers such as described in Hermanson, mentioned further above.Additionally, as noted above, the moieties of the elongated body of thetether, or the elongated tag of the nucleotide being acted upon, orboth, can be individual short nucleotide sequences that interact withone another. These moieties can be non-interacting with polymerase suchas RNA, PNA or LNA labels, morpholinos, or enantiomeric DNA, forexample. The moiety need not be formed of the same polymer as otherportions of the elongated body of the tether or the elongated tag of thenucleotide being acted upon. Elongated tags can be readily attached tothe gamma phosphate of nucleotides, as is well known in the art.Additionally, in one illustrative embodiment, that isoG and isoC basescan be used on the nucleotide elongated tags, or on the tether, or both,so as to inhibit hybridization of the elongated tags or tether with theDNA being sequenced. Additionally, other schemes can be used to inducesecondary structure in the tether to shorten it, such as a hairpin.

Additionally, note that the tether can include multiple moieties, eachof which respectively interacts with a moiety attached to a given typeof nucleotide. The interaction between the moiety of the nucleotide withthe corresponding moiety of the tether can move the reporter region ofthe tether by a corresponding amount that facilitates identification ofthe corresponding nucleotide via a signal, e.g., via a current or fluxthrough the aperture of the pore.

In one nonlimiting, illustrative embodiment, the pore includes MspA,which can provide a satisfactory separation of nucleotide-specificcurrents or fluxes, e.g., a 3.5-fold greater separation ofnucleotide-specific currents or fluxes as compared to alpha-hemolysin.However, it should be appreciated that alpha-hemolysin or other types ofpores suitably can be used with the present compositions, systems, andmethods.

Additionally, note that in embodiments in which a voltage is appliedacross the pore and movements of the reporter region are measured (e.g.,optically or electrically measured) via current or flux through thepore, the voltage can suitably be applied using either direct current(DC) or alternating current (AC). AC current can help to extendelectrode life, help to eject cleaved elongated tags from the pore ifthe tags become stuck, or can perform part of the work of pulling thetether up against the force of the applied voltage. Additionally, notethat a positively charged tether can be used with a reverse bias on thepore so as to inhibit the DNA being sequenced from being drawn into thepore. Additionally, note that a negatively-charged tether can bereverse-threaded through the pore (e.g., with the head region anchoredto the second side of the pore and the polymerase disposed adjacent tothe first side of the pore) with a reverse bias with negatively chargedelongated tags, so as to inhibit the elongated tags from jamming intothe constriction, and to inhibit the DNA being sequenced from enteringthe pore.

Additionally, a stochastic sensing method can be employed. In thisarrangement, an AC current can be used to move the hybridized duplexadjacent to the constriction, such as described in greater detail belowwith reference to FIGS. 18A-18E.

Exemplary Methods and Compositions for Detecting Action of a PolymeraseUpon a Nucleotide

It should be understood that alternative methods and compositions can beused to detect action of a polymerase upon a nucleotide. For example,FIG. 15 illustrates an alternative method for detecting action of apolymerase upon a nucleotide using a composition including a tetheranchored to or adjacent to a nanopore. For example, a composition caninclude a nanopore including a first side, a second side, and anaperture extending through the first and second sides; and a permanenttether including a head region, a tail region, and an elongated bodydisposed therebetween. The head region can be anchored to or adjacent tothe first side or second side of the nanopore, and the elongated bodycan include a moiety. A polymerase can be disposed adjacent to the firstside of the nanopore. The composition also includes a first nucleotideincluding a first elongated tag. The first elongated tag includes afirst moiety that interacts with the moiety of the tether responsive tothe polymerase acting upon the first nucleotide.

For example, method 1500 illustrated in FIG. 15 includes providing acomposition including a nanopore, a permanent tether, and a polymerasedisposed adjacent to the nanopore (step 1501). For example, FIG. 16schematically illustrates an exemplary composition including a tetheranchored to a nanopore and configured for use in detecting action of apolymerase upon a nucleotide. In the exemplary embodiment illustrated inFIG. 16, the composition can include nanopore 1600, permanent tether1610, and polymerase 1650. Nanopore 1600 includes first side 1601,second side 1602, aperture 1603 extending through sides 1601 and 1602,and optionally also includes constriction 1604. Permanent tether 1610includes head region 1611, tail region 1612, and elongated body 1613disposed therebetween. Polymerase 1650 is disposed adjacent to firstside 1601 of nanopore 1600. For example, polymerase 1650 can be incontact with first side 1601 of nanopore 1600, and optionally can beanchored to or adjacent to the first side of nanopore 1600 via anysuitable chemical bond, protein-protein interaction, or any othersuitable attachment that is normally irreversible. In the embodimentillustrated in FIG. 16, head region 1611 of tether 1610 is attached to,e.g., anchored to, first side 1601 of nanopore 1600, via any suitablechemical bond, protein-protein interaction, or any other suitableattachment that is normally irreversible.

Head region 1611 can be attached to any suitable portion of nanopore1600 that places elongated tag 1613 sufficiently close to polymerase1650 so as to interact with elongated tags of respective nucleotidesthat can be acted upon by polymerase 1650. For example, nucleotide 1630can include an elongated tag 1631 including moiety 1632 that interactswith tether 1610. In an illustrative embodiment, elongated tag 1613 oftether 1610 can include a moiety 1615 with which moiety 1632 of tag caninteract. Tail region 1612 can extend freely toward the second side ofthe nanopore, and can be disposed either on the first side of thenanopore, such as described below with reference to FIGS. 17A-17B, orcan be disposed on or beyond the second side of the nanopore, such asdescribed above with reference to FIGS. 7A-7B, or can be movable betweenthe first and second sides of the nanopore, such as described below withreference to FIGS. 19-20B. Optionally, tail region 1612 can be attachedto another member in a manner such as described with reference to FIGS.1I and 1M, which other member optionally can be disposed within aperture1603. Note that polymerase 1650 or nucleotide 1630, or both, can be, butneed not necessarily be, considered to be part of the inventivecomposition, but instead can be considered to be in contact with acomposition that includes nanopore 1600 and permanent tether 1610.

Referring again to FIG. 15, method 1500 includes acting upon anucleotide with the polymerase (step 1502). For example, FIG. 16schematically illustrates binding of nucleotide 1630 by polymerase 1650,but it should be understood that polymerase 1650 can act upon nucleotidein a variety of ways, e.g., by adding nucleotide 1630 to apolynucleotide, excising nucleotide 1630 from an existingpolynucleotide, or sampling nucleotide 1630, e.g., transientlyinteracting with nucleotide 1630 without binding it. Method 1500illustrated in FIG. 15 also includes interacting a moiety of thenucleotide with the tether (step 1503). For example, in the embodimentillustrated in FIG. 16, polymerase 1650 acting upon nucleotide 1630 canbring moiety 1632 of nucleotide 1630 into sufficiently close proximityto moiety 1615 that the moieties interact with one another, e.g., bondwith one another. Such an interaction can be reversible, e.g., caninclude formation of a hydrogen bond, ionic bond, dipole-dipole bond,London dispersion forces, reversible covalent bond, or any suitablecombination thereof

Referring again to FIG. 15, method 1500 also can include detecting theinteraction of the moiety with the tether in any suitable manner. Forexample, the elongated tag of the nucleotide can include a reporterregion, and method 1500 can include detecting the presence of thereporter region within the aperture of the nanopore (step 1504). Forexample, FIGS. 17A-17B schematically illustrate a composition includinga tether anchored to or adjacent to a nanopore and configured for use indetecting action of a polymerase upon a nucleotide including anelongated tag including a reporter region. As illustrated in FIG. 17A,the composition can include nanopore 1700, including first side 1701,second side 1702, aperture 1703 extending through the first and secondsides, and optional constriction 1704; permanent tether 1710 includinghead region 1711 anchored to first side 1701 of nanopore 1700, tailregion 1712 disposed on first side 1702 of nanopore 1700, and elongatedbody 1713 that includes moiety 1715 but lacks a reporter region; andnucleotide 1730 including elongated tag 1731 that includes moiety 1732and reporter region 1734.

As illustrated in FIG. 17B, an interaction between moiety 1732 ofnucleotide 1730 and moiety 1715 of tether 1710 can dispose reporterregion 1734 within aperture 1703. It should be appreciated that thedisposition of reporter region 1734 within aperture 1703 can bedetectable in any suitable manner. For example, the composition can bein operable communication with a measurement circuit such as describedabove with reference to FIG. 2A or FIG. 2C. The measurement circuit canbe configured to detect the disposition of reporter region 1734 withinaperture 1703. In one illustrative embodiment, nanopore 1700, tether1710, polymerase 1750, and nucleotide 1730 can be immersed in aconductive fluid, e.g., an aqueous salt solution. A measurement circuitconfigured analogously to measurement circuit 230 illustrated in FIG. 2Aor measurement circuit 240 illustrated in FIG. 2C can be incommunication with first and second electrodes and can be configured toapply a voltage between those electrodes so as to apply a voltage acrossnanopore 1700. The measurement circuit further can be configured to usethe electrodes to measure the magnitude of a current or flux throughaperture 1703. Reporter region 1734 can have a different electrical orflux blockade property than some or all other regions of elongated tag1731. For example, reporter region 1734 can include an electrostaticcharge, while some or all other regions of elongated tag 1731 caninclude a different electrostatic charge, or can be uncharged (e.g., canbe electrically neutral). Or, for example, reporter region 1734 can beuncharged, while some or all other regions of elongated body 1731 caninclude an electrostatic charge. The magnitude of the current or fluxthrough aperture 1703 can measurably change responsive to disposition ofreporter region 1734 within aperture 1703, and the time period for sucha measurable change in the current or flux can be based on the durationof the interaction between moieties 1715 and 1732, which in turn can bebased on the duration of the action of polymerase 1750 upon nucleotide1730. In one illustrative, nonlimiting example, elongated body 1731includes a polynucleotide that includes one or more abasic nucleotidesthat define reporter region 1734.

In one illustrative embodiment, the formation of a duplex can bemonitored using duplex interrupted sequencing such as described inDerrington et al., “Nanopore DNA sequencing with MspA,” Proc. Natl.Acad. Sci. USA, 107:16060-16065 (2010), the entire contents of which areincorporated by reference herein. The present system can uses an ACdriving voltage whose temporal period is on the same order of magnitudeas the time to duplex formation, which can be expected to besignificantly shorter than the polymerase catalytic incorporation eventbeing measured. See also PCT Publication No. WO2011/106459 to Gundlachet al., the entire contents of which are incorporated by referenceherein.

The action of polymerase 1750 upon nucleotide 1730 can be individuallyidentifiable based on a measured (e.g., optically or electricallymeasured) magnitude or time duration, or both, of a signal generated bysuch a system. For example, the action of polymerase 1750 uponnucleotide 1730 can cause interaction between moieties 1715 and 1732,which in turn causes reporter region 1734 to become disposed at a firstlocation within aperture 1703, and the presence of reporter region 1734at the first location causes the signal to have a first magnitude. Assuch, the signal having the first magnitude correlates to the action ofpolymerase 1750 upon nucleotide 1730 having occurred.

Note that in some embodiments, the respective lengths of elongated body1713 and elongated tag 1731, the respective locations of moieties 1715and 1732, and the respective location of reporter region 1734 areco-selected so as to inhibit the application of force to nucleotide 1730while the nucleotide is being acted upon by polymerase 1750, and thus toinhibit or preclude such a force from modifying the performance of thepolymerase. In one illustrative embodiment, the interaction betweenmoiety 1715 and moiety 1713 forms a duplex. The length of elongated body1713 can be selected such that the elongated body substantially does notextend through the location at which reporter region 1734 is to bedisposed. The length of elongated tag 1731 can be selected so as toextend through the location at which reporter region is to be disposed,while providing additional slack such that elongated tag 1731 need notbe pulled taut in order to dispose reporter region 1734 at the location.In some embodiments, the respective location of moiety 1715 alongelongated body 1713 of tether 1710 and the respective location of moiety1732 along elongated tag 1731 of nucleotide 1730 are co-selected so asto provide the additional slack in elongated tag 1731 at a locationbetween the duplex of 1715, 1732 and polymerase 1750. Accordingly, theanchoring of head region 1711 to pore 1700 can inhibit movement of theduplex 1715, 1732 through aperture 1703, and can absorb forces thatotherwise may have been applied to nucleotide 1730 via elongated tag1731. Additionally, reporter region 1734 can be disposed at a suitablelocation along elongated body 1731 so as to be disposed at a suitablelocation within aperture 1730 to facilitate detection of the reporterregion when moieties 1715 and 1732 interact with one another. In oneexemplary embodiment, reporter region 1734 is disposed at a suitablelocation along elongated body 1731 so as to be disposed within, oradjacent to, constriction 1704 of nanopore 1700 when moieties 1715 and1732 interact with one another responsive to action of polymerase 1750.

Other methods of detecting the action of a polymerase 1750 upon anucleotide suitably can be used. For example, method 1500 alternativelycan include changing an applied voltage across the nanopore aperture(step 1505), disposing a reporter region of a tether at a locationwithin the aperture responsive to the change in applied voltage (step1506), and detecting the presence of the reporter region of the tetherat the location within the aperture (step 1507). For example, FIGS.18A-18D schematically illustrate a composition including a tetheranchored to or adjacent to a nanopore and configured for use indetecting action of a polymerase upon a nucleotide using a tetheranchored to or adjacent to a nanopore responsive to a change inelectrical potential across the nanopore, and FIG. 18E illustrates anexemplary signal that can be generated during use of such a composition.

The composition illustrated in FIG. 18A includes nanopore 1800 includingfirst side 1801, second side 1802, aperture 1803 extending through thefirst and second sides, and constriction 1804 disposed between the firstand second sides; permanent tether 1810 including a head region (notspecifically labeled) anchored to first side 1801 of nanopore 1800, atail region (not specifically labeled) that is movable between firstside 1801 and second side 1802 of nanopore 1800, and an elongated body(not specifically labeled) that includes reporter region 1814 and moiety1815; and nucleotide 1830 including an elongated tag (not specificallylabeled) that includes moiety 1832 but lacks a reporter region. Asillustrated in FIG. 18A, an interaction between moiety 1832 ofnucleotide 1830 and moiety 1815 of tether 1810 can dispose reporterregion 1814 at a predetermined location relative to the moiety 1832.Optionally, more than one reporter region can be provided, e.g., atleast two, or three, or four, or five, or more than five reporterregions. Additionally, moiety 1815 can be located at any suitableposition along elongated tag 1813, e.g., can be located between headregion 1811 and reporter region 1814 and adjacent to reporter region1814 such as illustrated in FIG. 18A, or can be adjacent to head region1811, adjacent to tail region 1812, or between tail region 1812 andreporter region 1814.

It should be appreciated that the disposition of reporter region 1814 atthe predetermined location relative to moiety 1832 can be detectable inany suitable manner. For example, the composition can be in operablecommunication with a measurement circuit such as described above withreference to FIG. 2A or FIG. 2C. The measurement circuit can beconfigured to detect the position of reporter region 1814 relative tomoiety 1832. In one illustrative embodiment, nanopore 1800, tether 1810,polymerase 1850, and nucleotide 1830 can be immersed in a conductivefluid, e.g., an aqueous salt solution. A measurement circuit configuredanalogously to measurement circuit 230 illustrated in FIG. 2A ormeasurement circuit 240 illustrated in FIG. 2C can be in communicationwith first and second electrodes and can be configured to apply a firstvoltage between those electrodes so as to apply a voltage acrossnanopore 1800, as represented by the “+” and “−” signs illustrated inFIG. 18A, and to use the electrodes to measure the magnitude of acurrent or flux through aperture 1803 at the first voltage. The portionof FIG. 18E immediately below FIG. 18A illustrates an exemplary currentor flux through aperture 1803 at the first voltage. Reporter region 1814can have a different electrical or flux blockade property than some orall other regions of the elongated body of the tether (not specificallylabeled). For example, reporter region 1814 can include an electrostaticcharge, while some or all other regions of elongated body can include adifferent electrostatic charge, or can be uncharged (e.g., can beelectrically neutral). Or, for example, reporter region 1814 can beuncharged, while some or all other regions of the elongated body caninclude an electrostatic charge. In one illustrative, nonlimitingexample, the elongated body of the tether includes a polynucleotide thatincludes one or more abasic nucleotides that define reporter region1814. The magnitude of the current or flux through aperture 1803 canmeasurably change responsive to the relative location of reporter region1814 within aperture 1803, and such relative location can be based uponthe applied voltage and on the location of reporter region 1814 relativeto moiety 1832, which in turn can be based on the action of polymerase1850 upon nucleotide 1830.

More specifically, the measurement circuit further can be configured tochange the applied voltage across nanopore 1800 to a second voltage,e.g., by reversing the applied voltage such as represented by thereversal of the “+” and “−” signs such as illustrated in FIG. 18B. Sucha change in applied voltage can cause movement of interacting moieties1815, 1832 within aperture 1803 of nanopore 1800. For example, asillustrated in FIG. 18B, the change in applied voltage can moveinteracting moieties 1815, 1832 adjacent to constriction 1804, and candispose reporter region 1814 adjacent to or within constriction 1804.The measurement circuit can be configured to use the electrodes tomeasure the magnitude of a current or flux through aperture 1803 at thesecond voltage. The portion of FIG. 18E immediately below FIG. 18Billustrates an exemplary current or flux through aperture 1803 at thesecond voltage. It can be seen that the current or flux at the firstvoltage is different than the current or flux at the second voltage, andsuch current or flux can be based upon the second voltage and on thelocation of reporter region 1814 relative to moiety 1832, which in turncan be based on the action of polymerase 1850 upon nucleotide 1830.

The action of polymerase 1850 upon nucleotide 1830 can be individuallyidentifiable based on a measured (e.g., optically or electricallymeasured) magnitude or time duration, or both, of a signal generated bysuch a system. For example, the action of polymerase 1850 uponnucleotide 1830 can cause interaction between moieties 1815 and 1832,which in turn causes reporter region 1814 to become disposed at a firstlocation relative to moiety 1832, and the presence of reporter region1814 at the first location causes the signal, e.g., current or fluxthrough aperture 1803, to have a first magnitude. As such, the signalhaving the first magnitude correlates to the action of polymerase 1850upon nucleotide 1830 having occurred. Note that a duplex formed betweenmoiety 1815 and moiety 1832 can be sufficiently large as to inhibitmovement of the duplex through the constriction, e.g., under the secondvoltage.

As illustrated in FIG. 18C, in some embodiments, continued applicationof the second voltage can cause moiety 1815 to dissociate from moiety1832. Such dissociation can be considered to “interrupt” a duplex formedbetween moiety 1815 and moiety 1832. In some embodiments, reporterregion 1814 or moiety 1815, or both, can move through constriction 1804so as to be disposed on second side 1802 of nanopore 1800. The portionof FIG. 18E immediately below FIG. 18C illustrates an exemplary currentor flux through aperture 1803 at the second voltage, followingdissociation of moiety 1815 from moiety 1832. Moiety 1832 can beconfigured so as to remain disposed on the first side of nanopore 1800even if moiety 1815 becomes disposed on the second side of nanopore1800, so as to temporarily inhibit interaction between moieties 1815 and1832. As illustrated in FIG. 18D, following such dissociation, thevoltage applied across aperture 1803 can again be changed, e.g., can bechanged back to the first voltage, responsive to which moieties 1815 and1832 can interact with one another. The portion of FIG. 18E immediatelybelow FIG. 18D illustrates an exemplary current or flux through aperture1803 at the first voltage, following interaction of moiety 1815 frommoiety 1832.

Note that in some embodiments, the respective lengths of the elongatedbody of the tether and the elongated tag of the nucleotide, therespective locations of moieties 1815 and 1832, and the respectivelocation of reporter region 1814 are co-selected so as to inhibit theapplication of force to nucleotide 1830 while the nucleotide is beingacted upon by polymerase 1850, and thus to inhibit or preclude such aforce from modifying the performance of the polymerase. In oneillustrative embodiment, the interaction between moiety 1815 and moiety1832 forms a duplex. The length of the elongated body of the tether, andthe location of moiety 1815 along the elongated body, can be co-selectedsuch that moiety 1815 can be extended through constriction 1804responsive to an appropriate applied voltage, e.g., so as to causedissociation between moiety 1815 and moiety 1832. The length of theelongated tag of the nucleotide, and the location of moiety 1832 alongthe elongated tag, can be co-selected so as to provide additional slacksuch that elongated tag need not be pulled taut in order to disposereporter region 1814 adjacent to constriction 1804 under the secondapplied voltage. The size of the duplex 1815, 1832 can inhibit movementof the duplex through constriction 1804, and can shield the nucleotidefrom forces that otherwise may have been applied to nucleotide 1830 viaelongated tag 1831. Additionally, the relative locations of reporterregion 1814 and moieties 1815 and 1832 can be co-selected so as todispose reporter region 1814 at a suitable location relative toconstriction 1804 under the second voltage so as to facilitate detectionof the reporter region when moieties 1815 and 1832 interact with oneanother. In one exemplary embodiment, reporter region 1814 is disposedat a suitable location along elongated body 1831 so as to be disposedwithin, or adjacent to, constriction 1804 of nanopore 1800 when moieties1815 and 1832 interact with one another responsive to action ofpolymerase 1850.

As yet another alternative method of detecting the action of apolymerase upon a nucleotide, method 1500 alternatively can includedetecting the movement of a reporter region of a tether within anaperture (step 1508). Exemplary compositions for detecting the movementof a reporter region of a tether in association with a nucleotide actingupon a polymerase are described further above with reference to FIGS.7A-14.

Note that following any of steps 1507, 1507, or 1508, method 1500further can include releasing the moiety of the nucleotide from thetether, in a manner analogous to that described above with reference toFIGS. 7A-14 (step not specifically illustrated in FIG. 15).Additionally, as described in greater detail below with reference toFIGS. 19A-20B, or above with reference to FIGS. 7A-14, the presentcompositions and methods can be used to individually detect the actionof polymerases on different nucleotides.

Sequencing by Synthesis Using Exemplary Methods and Compositions Basedon Detecting Action of Polymerases Upon Nucleotides

It should be appreciated that method 1500 illustrated in FIG. 15suitably can be used to detect action of a polymerase upon any type ofnucleotide having a suitable moiety attached thereto. In illustrativeembodiments described below with reference to FIGS. 18-22F, method 1500can be used to detect a polymerase's action upon a nucleotide and theuse thereof to sequence a first polynucleotide by synthesizing a secondpolynucleotide that is complementary to the first nucleotide, e.g.,using “sequencing by synthesis” (SBS).

FIGS. 19A-19B schematically illustrate a composition including a tetheranchored to or adjacent to a nanopore and configured for use indetecting action of a polymerase upon a nucleotide including anelongated tag including a reporter region. The nanopore includesbiological pore 1905, which can be disposed in a barrier (notspecifically illustrated), e.g., a membrane of biological origin such asa lipid bilayer, or a solid state membrane. Biological pore 1905includes aperture 1903 and constriction 1904. The permanent tetherincludes head region 1911, elongated body 1913, and moiety 1915.Polymerase 1950 is disposed adjacent to, and in contact with, biologicalpore 1905, and optionally can be anchored to biological pore 1905 via aphysical or chemical linkage (e.g., using click chemistry or acysteine-maleimide bond). Polymerase 1950 is configured to receive atemplate polynucleotide 1970, e.g., circular or linear ssDNA to besequenced, to synthesize a polynucleotide 1960 having a complementarysequence to that of the ssDNA by sequentially receiving, binding, andadding nucleotides to the polynucleotide in accordance with the sequenceof the ssDNA. Head region 1911 of the tether can be anchored to anysuitable portion of biological pore 1905 that places moiety 1915sufficiently close to polymerase 1950 so as to interact withcorresponding moieties of nucleotides that can be bound by polymerase1950. For example, as illustrated in FIG. 19B, nucleotide 1930 caninclude an elongated tag 1931 including moiety 1932 that interacts withmoiety 1915 of the tether, as well as reporter region 1934 configured tobe disposed through aperture 1903 of nanopore 1905.

In one example, a voltage can be applied across the nanopore 1905, e.g.,using measurement circuit 230 and electrodes 231, 232 such as describedfurther above with reference to FIG. 2A or measurement circuit 240 andelectrodes 241, 242 such as described further above with reference toFIG. 2C. Reporter region 1914 or elongated body 1913 optionally includesan electrostatic charge that, responsive to the applied voltage, causestail region 1912 of elongated body 1913 to extend toward second side1902 of nanopore 1905. Additionally, elongated tag 1931 of nucleotide1930 includes an electrostatic charge that, responsive to the appliedvoltage, causes end region 1933 of tag 1931 to pass through constriction1904 such that reporter region 1934 is disposed within or adjacent toconstriction 1904. Responsive to polymerase 1950 binding nucleotide1930, moiety 1932 of nucleotide 1930 can reversibly bond to moiety 1915of tether 1932, which can dispose reporter region 1934 within oradjacent to constriction 1904. As a result, the binding of nucleotide1930 by polymerase 1950 can be translated or transduced into ameasurable change in current or flux through constriction 1904, whichalso can be referred to as a blockade current or flux. Additionally, theforce exerted on tether by the applied voltage is expected to pull onthe pore via moiety 1915 rather than on the polymerase, and thus is notexpected to significantly disrupt polymerase activity.

In one illustrative embodiment, moiety 1915 includes a firstoligonucleotide, and moiety 1932 includes a second oligonucleotide thatis complementary to the first oligonucleotide, e.g., that hybridizes tothe first oligonucleotide. The hybridization of the secondoligonucleotide to the first oligonucleotide can cause reporter region1934 to become disposed within or adjacent to constriction 1904. Thebinding of nucleotide 1930 can be individually detected based on ameasured (e.g., optically or electrically measured) magnitude or timeduration, or both, of a current or flux through constriction 1904. Forexample, FIG. 19B schematically illustrates an exemplary nucleotide1930, e.g., T, including an elongated tag 1931 including anoligonucleotide moiety 1932 that can be attached to the gamma phosphateof the nucleotide 1930, e.g., via a delta phosphate linkage.Oligonucleotide moiety 1932 can include any suitable sequence ofnucleotides selected to hybridize to a corresponding sequence ofnucleotides within moiety 1915 of the tether. For example,oligonucleotide moiety 1932 illustrated in FIG. 19B can include theexemplary sequence TACG, and moiety 1915 can include the complementarysequence ATGC. In a manner analogous to that described above withreference to FIGS. 8A-14, the action of polymerase 1905 upon nucleotide1930 can maintain moiety 1932 in relatively close proximity to moiety1915 of the tether, resulting in a transient increase in the localconcentration of oligonucleotide moiety 1932 that can inducehybridization between moieties 1932 and 1915 preferentially to moietiesthat are attached to nucleotides not presently being acted upon bypolymerase 1905. The resulting hybridization causes disposition ofreporter region 1934 adjacent to or within constriction 1904. Polymerase1950 can release elongated tag 1931 upon incorporating nucleotide 1930into a polynucleotide, responsive to which moiety 1932 can dissociatefrom moiety 1915.

Each different type of nucleotide can include a corresponding elongatedtag that is attached to its gamma phosphate in a manner analogous tothat illustrated in FIG. 19B. For example, FIG. 19C schematicallyillustrates exemplary nucleotides including elongated tags that includerespective reporter regions and moieties that bond to an exemplarytether during use in detecting action of a nucleotide by a polymerasedisposed adjacent to a nanopore. As shown in FIG. 19C, A, T, C, and Gnucleotides can include respectively elongated tags that includedifferent reporter regions than one another, e.g., as respectivelyrepresented by the triangle, diamond, square, and circle. For furtherinformation about reporter regions that can be attached to nucleotidesso as to permit distinguishing the nucleotides from one another, seeU.S. Pat. No. 8,652,779 to Turner et al., the entire contents of whichare incorporated by reference herein. Additionally, elongated tagsinclude moieties that can be suitably selected so as to hybridize to acorresponding moiety of the permanent tether. However, the moieties neednot be different than one another, and indeed can be the same as oneanother because the nucleotides can be distinguishable from one anotherbased on differences between their respective reporter regions. In oneillustrative embodiment, the moieties have lengths of 5 to 8nucleotides. It is expected that the melting temperatures of duplexesbetween moieties on incorporating nucleotides and moieties on the tetherto be significantly more stable than an otherwise identical pair offreely diffusing oligonucleotides because the tether and theincorporating nucleotide are held in relatively fixed position relativeto another, causing an effective increase in the local concentrations ofthe moieties, as discussed above with reference to FIGS. 8A-14.

Other compositions suitably can be used to perform sequencing bysynthesis based on detection action of a polymerase upon nucleotides.For example, FIGS. 20A-20D schematically illustrate a compositionincluding a tether anchored to or adjacent to a nanopore and configuredfor use in detecting action of a polymerase upon a first nucleotideusing a tether anchored to or adjacent to a nanopore responsive to achange in electrical potential across the nanopore, and FIG. 20Eillustrates an exemplary signal that can be generated during use of sucha composition.

The composition illustrated in FIG. 20A includes nanopore 2000 includingfirst side 2001, second side 2002, aperture 2003 extending through thefirst and second sides, and constriction 2004 disposed between the firstand second sides; a permanent tether (not specifically labeled)including a head region (not specifically labeled) anchored to secondside 2002 of nanopore 2000, a tail region (not specifically labeled)that is movable between first side 2001 and second side 2002 of nanopore2000, and an elongated body (not specifically labeled) that includes aplurality of reporter regions 2014, 2024, 2034, and moiety 2015; andnucleotide 2030 including an elongated tag (not specifically labeled)that includes moiety 2032 but lacks a reporter region. As illustrated inFIG. 20A, an interaction between moiety 2032 of nucleotide 2030 andmoiety 2015 of tether 2010 can dispose each reporter region 2014, 2024,2034 at a predetermined location relative to the moiety 2032. Anysuitable number of reporter regions can be provided, e.g., at least tworeporter regions, three reporter regions, four reporter regions, fivereporter regions, or more than five reporter regions. Additionally,moiety 2015 can be located at any suitable position along elongated tag2013, e.g., can be located between and adjacent to each of tail region2012 and first reporter region 2014 such as illustrated in FIG. 20A, orcan be adjacent to head region 2011, or between head region 2011 andthird reporter region 2034, or between any of reporter regions 2014,2024, or 2034.

It should be appreciated that the relative position of moiety 2032 andone or more of the reporter regions, e.g., reporter region 2014 can bedetectable in any suitable manner. For example, the composition can bein operable communication with a measurement circuit such as describedabove with reference to FIG. 2A or FIG. 2C. The measurement circuit canbe configured to detect the position of reporter region 2014 relative tomoiety 2032. In one illustrative embodiment, nanopore 2000, tether 2010,polymerase 2050, and nucleotide 2030 can be immersed in a conductivefluid, e.g., an aqueous salt solution. A measurement circuit configuredanalogously to measurement circuit 230 illustrated in FIG. 2A ormeasurement circuit 240 illustrated in FIG. 2C can be in communicationwith first and second electrodes and can be configured to apply a firstvoltage between those electrodes so as to apply a voltage acrossnanopore 2000, as represented by the “+” and “−” signs illustrated inFIG. 20A, and to use the electrodes to measure the magnitude of acurrent or flux through aperture 2003 at the first voltage. The portionof FIG. 20E immediately below FIG. 20A illustrates an exemplary currentor flux through aperture 2003 at the first voltage. Reporter region 2014can have a different electrical or flux blockade property than some orall other regions of the elongated body of the tether (not specificallylabeled), as well as than some or all other reporter regions 2024, 2034.The magnitude of the current or flux through aperture 2003 canmeasurably change responsive to the relative location of reporter region2014 within aperture 2003, and such relative location can be based uponthe applied voltage and on the location of reporter region 2014 relativeto moiety 2032, which in turn can be based on the action of polymerase2050 upon nucleotide 2030.

More specifically, the measurement circuit further can be configured tochange the applied voltage across nanopore 2000 to a second voltage,e.g., by reversing the applied voltage such as represented by thereversal of the “+” and “−” signs such as illustrated in FIG. 20B. Sucha change in applied voltage can cause movement of interacting moieties2015, 2032 within aperture 2003 of nanopore 2000. For example, asillustrated in FIG. 20B, the change in applied voltage can moveinteracting moieties 2015, 2032 adjacent to constriction 2004, and candispose reporter region 2014 adjacent to or within constriction 2004selectively relative to reporter regions 2024, 2034. The measurementcircuit can be configured to use the electrodes to measure the magnitudeof a current or flux through aperture 2003 at the second voltage. Theportion of FIG. 20E immediately below FIG. 20B illustrates an exemplarycurrent or flux through aperture 2003 at the second voltage. It can beseen that the current or flux at the first voltage is different than thecurrent or flux at the second voltage, and such current or flux can bebased upon the second voltage and on the location of reporter region2014 relative to moiety 2032, which in turn can be based on the actionof polymerase 2050 upon nucleotide 2030.

The action of polymerase 2050 upon nucleotide 2030 can be individuallyidentifiable based on a measured (e.g., optically or electricallymeasured) magnitude or time duration, or both, of a signal generated bysuch a system. For example, the action of polymerase 2050 uponnucleotide 2030 can cause interaction between moieties 2015 and 2032,which in turn causes reporter region 2014 to become disposed at a firstlocation relative to moiety 2032, and the presence of reporter region2014 at the first location causes the signal, e.g., current or fluxthrough aperture 2003, to have a first magnitude. As such, the signalhaving the first magnitude correlates to the action of polymerase 2050upon nucleotide 2030 having occurred. Note that a duplex formed betweenmoiety 2015 and moiety 2032 can be sufficiently large as to inhibitmovement of the duplex through the constriction, e.g., under the secondvoltage.

As illustrated in FIG. 20C, in some embodiments, continued applicationof the second voltage can cause moiety 2015 to dissociate from moiety2032. Such dissociation can be considered to “interrupt” a duplex formedbetween moiety 2015 and moiety 2032. In some embodiments, reporterregion 2014 or moiety 2015, or both, can move through constriction 2004so as to be disposed on second side 2002 of nanopore 2000. The portionof FIG. 20E immediately below FIG. 20C illustrates an exemplary currentor flux through aperture 2003 at the second voltage, followingdissociation of moiety 2015 from moiety 2032. Moiety 2032 can beconfigured so as to remain disposed on the first side of nanopore 2000even if moiety 2015 becomes disposed on the second side of nanopore2000, so as to temporarily inhibit interaction between moieties 2015 and2032. As illustrated in FIG. 20D, following such dissociation, thevoltage applied across aperture 2003 can again be changed, e.g., can bechanged back to the first voltage, responsive to which moieties 2015 and2032 can interact with one another. The portion of FIG. 20E immediatelybelow FIG. 20D illustrates an exemplary current or flux through aperture2003 at the first voltage, following interaction of moiety 2015 frommoiety 2032.

As part of the action of polymerase 2050 upon nucleotide 2030,polymerase 2050 can, for example, cleave the elongated tag fromnucleotide 2030, causing dissociation of moiety 2032 from moiety 2015,and can add nucleotide 2030 to polynucleotide 2060 in accordance withthe sequence of template 2070. Polymerase 2050 then can act upon asecond nucleotide. For example, FIGS. 21A-21D schematically illustratethe composition of FIGS. 20A-20D configured for use in detecting actionof the polymerase upon a second nucleotide using the tether anchored toor adjacent to a nanopore responsive to a change in electrical potentialacross the nanopore, and FIG. 21E illustrates an exemplary signal thatcan be generated during use of such a composition. More specifically,FIG. 21 illustrates polymerase 2050 acting upon second nucleotide 2030′having second moiety 2032′. Second moiety 2032′ of second nucleotide2030′ interacts with moiety 2015 in a different manner than does moiety2032 of first nucleotide 2030.

For example, as illustrated in FIG. 21A, an interaction between moiety2032′ of second nucleotide 2030′ and moiety 2015 of the tether candispose each reporter region 2014, 2024, 2034 at a predeterminedlocation relative to the moiety 2032′ that is different than thepredetermined locations illustrated in FIG. 21A because moiety 2032′ isdifferent than moiety 2032, e.g., interacts with a different portion ofmoiety 2015 than does moiety 2023. The measurement circuit can beconfigured to detect the position of reporter region 2024 relative tomoiety 2032′ responsive to first and second applied voltages in a manneranalogous to that described above with reference to FIGS. 20A-20E. Forexample, the portion of FIG. 21E immediately below FIG. 21A illustratesan exemplary current or flux through aperture 2003 at the first voltage.Reporter region 2024 can have a different electrical or flux blockadeproperty than some or all other regions of the elongated body of thetether (not specifically labeled), as well as than some or all otherreporter regions 2014, 2034.

The magnitude of the current or flux through aperture 2003 canmeasurably change responsive to the relative location of reporter region2014 within aperture 2003, and such relative location can be based uponthe applied voltage and on the location of reporter region 2014 relativeto moiety 2032′, which in turn can be based on the action of polymerase2050 upon nucleotide 2030′. More specifically, the measurement circuitfurther can be configured to change the applied voltage across nanopore2000 to a second voltage, e.g., by reversing the applied voltage such asrepresented by the reversal of the “+” and “−” signs such as illustratedin FIG. 21B. Such a change in applied voltage can cause movement ofinteracting moieties 2015, 2032′ within aperture 2003 of nanopore 2000.For example, as illustrated in FIG. 21B, the change in applied voltagecan move interacting moieties 2015, 2032′ adjacent to constriction 2004,and can dispose reporter region 2024 adjacent to or within constriction2004 selectively relative to reporter regions 2014, 2034. Themeasurement circuit can be configured to use the electrodes to measurethe magnitude of a current or flux through aperture 2003 at the secondvoltage. The portion of FIG. 21E immediately below FIG. 21B illustratesan exemplary current or flux through aperture 2003 at the secondvoltage. It can be seen that the current or flux at the first voltage isdifferent than the current or flux at the second voltage, and suchcurrent or flux can be based upon the second voltage and on the locationof reporter region 2024 relative to moiety 2032′, which in turn can bebased on the action of polymerase 2050 upon nucleotide 2030′. Forexample, it can be seen that the current or flux illustrated in FIG. 21Eas corresponding to FIG. 21B is greater than the current or fluxillustrated in FIG. 20E as corresponding to FIG. 20B, because reporterregion 2024 has a measurably different characteristic than does reporterregion 2014. As such, the magnitude of the signal, e.g., current orflux, correlates to the particular type of nucleotide upon whichpolymerase 2050 is acting.

As illustrated in FIG. 21C, in some embodiments, continued applicationof the second voltage can cause moiety 2015 to dissociate from moiety2032′ in a manner analogous to that described above with reference toFIG. 20C. The portion of FIG. 21E immediately below FIG. 21C illustratesan exemplary current or flux through aperture 2003 at the first voltage,following interaction of moiety 2015 from moiety 2032′. As illustratedin FIG. 21D, following such dissociation, the voltage applied acrossaperture 2003 can again be changed, e.g., can be changed back to thefirst voltage, responsive to which moieties 2015 and 2032′ can interactwith one another in a manner described above with reference to FIG. 20D.The portion of FIG. 21E immediately below FIG. 21D illustrates anexemplary current or flux through aperture 2003 at the first voltage,following interaction of moiety 2015 from moiety 2032′.

Note that in some embodiments, the respective lengths of the elongatedbody of the tether and the elongated tag of the nucleotide, therespective locations of moieties 2015 and 2032 and 2032′, and therespective locations of reporter regions 2014, 2024, and 2034 areco-selected so as to inhibit the application of force to nucleotide 2030or 2030′ while the nucleotide respectively is being acted upon bypolymerase 2050, and thus to inhibit or preclude such a force frommodifying the performance of the polymerase, as well as to permitdifferent nucleotides to be individually distinguishable from oneanother. In one illustrative embodiment, the interaction between moiety2015 and moiety 2032 or 2032′ forms a duplex. The length of theelongated body of the tether, and the location of moiety 2015 along theelongated body, can be co-selected such that moiety 2015 can be extendedthrough constriction 2004 responsive to an appropriate applied voltage,e.g., so as to permit interaction between moiety 2015 and moiety 2032 ormoiety 2032′. The length of the elongated tag of the nucleotide, and thelocation of moiety 2032 or 2032′ along the elongated tag, can beco-selected so as to provide additional slack such that elongated tagneed not be pulled taut in order to respectively dispose one of reporterregions 2014, 2024, or 2034 within or adjacent to constriction 2004under the second applied voltage. The size of the duplex 2015, 2032 or2015, 2032′ can inhibit movement of the duplex through constriction2004, and can shield the respective nucleotide 2030, 2030′ from forcesthat otherwise may have been applied to that nucleotide 2030 or 2030′via elongated tag 2031. Additionally, the relative locations of reporterregions 2014, 2024, and 2034 and moieties 2015 and 2032 and 2032′ can beco-selected so as to dispose one of those reporter regions at a suitablelocation relative to constriction 2004 under the second voltage so as tofacilitate detection of that reporter region when moieties 2015 and 2032or moieties 2015 and 2032′ respectively interact with one another. Inone exemplary embodiment, reporter region 2014 is disposed at a firstlocation along elongated body 2031 so as to be disposed within, oradjacent to, constriction 2004 of nanopore 2000 when moieties 2015 and2032 interact with one another responsive to action of polymerase 2050,reporter region 2024 is disposed at a second location along elongatedbody 2031 so as to be disposed within, or adjacent to, constriction 2004of nanopore 2000 when moieties 2015 and 2032′ interact with one anotherresponsive to action of polymerase 2050, and reporter region 2034 isdisposed at a suitable location along elongated body 2031 so as to bedisposed within, or adjacent to, constriction 2004 of nanopore 2000 whenmoiety 2015 interacts with a moiety of yet another nucleotide responsiveto action of polymerase 2050. Accordingly, it should be understood thatany suitable number of reporter regions can be provided so as to providedetectable signals corresponding to particular nucleotides being actedupon by the polymerase.

Note that the embodiment described further above with reference to FIGS.18A-18E, in which the head region of the tether is anchored to the firstside of the nanopore rather than to the second side of the nanopore, canbe used in a manner analogous to that of FIGS. 20A-21E so as toindividually identify nucleotides being acted upon by a polymerase.

Still other configurations suitably can be used. For example, FIGS.22A-22F schematically illustrate a composition including a tetheranchored adjacent to a nanopore and configured for use in detectingaction of a polymerase upon a first nucleotide using a change in appliedvoltage across the nanopore, according to some embodiments of thepresent invention.

More specifically, FIG. 22A illustrates a composition including nanopore2300 including first side 2201, second side 2202, aperture 2203extending through the first and second sides, and constriction 2204disposed between the first and second sides. Illustratively, nanopore2200 can include a biological pore, such as a MspA nanopore (e.g.,M2-NNN MspA mutant), disposed in a barrier, such as a membrane ofbiological origin (e.g., a lipid bilayer) or a solid state membrane. Thecomposition illustrated in FIG. 22A further includes tether 2210including head region 2211, tail region 2212, and elongated body 2213disposed therebetween. Head region 2211 is suitably anchored topolymerase 2250, e.g., using any suitable attachment provided herein orotherwise known in the art. Elongated body 2213 of tether 2210 caninclude a moiety 2214. Illustratively, elongated body 2213 can include apolynucleotide, and a first subset of the nucleic acids of thepolynucleotide can define moiety 2214. Additionally, tail region 2212can include at least one charged atom such that, based upon a voltagebeing applied across nanopore 2200 illustrated in FIG. 22A during step1, such voltage generates a first directional force F1 that causestranslocation of tail region 2212 through aperture 2203 and pastconstriction 2204 such that a portion of elongated tail 2213 becomesdisposed within aperture 2203 and tail region becomes disposed beyondsecond side 2202 of nanopore 2200 in a manner such as illustrated inFIG. 22B. For example, such voltage can be applied using a system suchas described herein with reference to FIGS. 2A-2C. Such directionalforce F1 also causes translocation of polymerase 2250 towards secondside 2202 of nanopore 2200 until polymerase 2250 comes to rest on oradjacent to first side 2201 of nanopore 2200 in a manner such asillustrated in FIG. 22B, preventing or inhibiting further movement ofpolymerase 2250 under directional force F1. Note that polymeraseoptionally can be partially disposed within aperture 2203 of nanopore2200.

The composition illustrated in FIG. 22A also can include another member2250′ to which tail region 2212 of tether 210 can attach in a manneranalogous to that described above with reference to FIG. 1M. Forexample, the composition illustrated in FIG. 22A can include one or morepolynucleotides 2250′ having a sequence that suitably can hybridize tocorresponding nucleic acids on elongated body 2213 or on tail region2212 of tether 2210. For example, as illustrated in FIG. 22B, underdirectional force F1 that is applied during step 1 (FIG. 22A) and cancontinue during step 2 (FIG. 22B), tail region 2212 becomes disposedbeyond second side 2202 of nanopore 2200 and becomes attached to, e.g.,hybridizes with member 2250′, e.g., a complimentary piece of DNA(“capture-DNA”) present adjacent to second side 2202 (e.g., on the transside) of nanopore 2200. The bond between tail region 2212 and member2250′, e.g., hybridization between one or more first nucleic acids oftail region 2212 and one or more second nucleic acids of member 2250′ soas to form a duplex 2212, 2250′, e.g., double stranded DNA, issufficiently strong so that upon application of a reverse directionalforce F2 (e.g., during step 3 illustrated in FIG. 22C), e.g., reversalof the voltage, the duplex inhibits separation of the polymerase fromthe nanopore and, as such, the polymerase remains captured at thenanopore. For example, duplex 2212, 2250′ can include a sufficientnumber of hybridized nucleic acids such that the duplex does notdissociate under application of force F2. Additionally, the duplex 2212,2250′ can be sufficiently large as to inhibit movement of the duplexthrough constriction 2204. Additionally, in some embodiments, thelateral dimensions of constriction 2204 of nanopore 2200 are selectedsuch that only a single elongated body 2213 of a single tether 2210 canbe disposed therethrough, thus assuring that only one polymerase 2250becomes captured at the nanopore.

In particular embodiments, a quality assessment step can be utilized toevaluate the nanopore or the capture of polymerase at the nanopore. Ananopore that is properly embedded in a membrane can produce acharacteristic current or flux pattern that is distinguishable from thecurrent or flux pattern that results when no nanopore is present in themembrane or when a nanopore is not fully functional. In the event that aquality assessment indicates that a nanopore is not properly embedded ina membrane, the steps used to load the nanopore can be repeated.

A polymerase that is properly captured by a nanopore can also produce acharacteristic current or flux pattern. For example, a bias voltage thatis applied to a nanopore that has captured a polymerase via a tether canproduce a current or flux pattern that is indicative of interactionbetween the nanopore aperture and signature bases in a nucleic acidtether. Bias voltages can be applied in opposite directions to determinewhether the tether has desired mobility in the nanopore lumen such thatsignature bases interact with the aperture as predicted. In the eventthat a quality assessment indicates that a polymerase has not beenproperly captured by a nanopore, the polymerase can be stripped, forexample by application of a strong reverse bias, and steps used tocapture the polymerase at the nanopore can be repeated.

In another optional quality assessment routine, a relatively largereverse bias voltage can be applied to the system to determine if thepolymerase and tether are removed from the nanopore. Typically, theduplex formed between member 2250′ and 2212 will be sufficiently strongto prevent removal of the tether. This quality assessment routine willindicate if this is the case. Similarly, bias voltages can be applied atthis stage and the resulting current or flux patterns detected todetermine if corking or uncorking occurs as set forth previously herein.In the event that a quality assessment indicates that a polymerase hasnot been captured by a nanopore with sufficient stability, steps used tocapture the polymerase at the nanopore can be repeated.

Several embodiments set forth herein relate to multiplex devices thatare loaded with multiple nanopores each of which is desired to attach toa polymerase. Quality assessment steps, such as those set forth above,can be carried out for the multiplex population. If a desired number offunctional nanopores have not been formed in a multiplex nanoporeapparatus or if the fractional loading is not sufficient, then theapparatus can be treated in bulk to repeat nanopore (or polymerase)loading. Optionally, the nanopores (or polymerases) can be removed priorto repeating the loading step, for example, if faulty nanopores orpolymerases are present. For example, repetition of loading (andoptionally removal of nanopores or polymerases) can be carried out ifthe multiplex apparatus is loaded at fewer than 90%, 75%, 50%, 30% orfewer of the expected sites.

At step 3 illustrated in FIG. 22C, the composition illustrated in FIG.22B further can be subjected to a reverse directional force F2, e.g.,reversal of the voltage relative to that of steps 1 and 2, based uponwhich polymerase 2250 can come out of contact with first side 2201 ofnanopore 2200, and can be contacted with sequencing primer 2280, targetsingle stranded DNA 2270 (target), and a plurality of nucleotides 2230,2230′, each of which includes a corresponding elongated tag 2231, 2231′including a corresponding moiety 2232, 2232′ that interacts with themoiety of tether 2213 responsive to polymerase 2250 acting upon thatnucleotide 2230 or 230′.

At step 4 illustrated in FIG. 22D, based upon the sequence of target2270,_polymerase 2250 acts upon first nucleotide 2230, based upon whichthe corresponding moiety 2232 of elongated tag 2231 of nucleotide 2230interacts with moiety 2214 of tether 2310. For example, polymerase 2250can preferentially bind first nucleotide 2230 relative to secondnucleotide 2230′ based upon first nucleotide 2230 being complementary toa next nucleotide in the sequence of target 2270. Additionally,elongated tag 2231 can include a first nucleotide sequence, and moiety2214 of elongated body 2213 can include a second nucleotide sequencethat is complementary to the first nucleotide sequence of elongated tag2231, such that the first nucleotide sequence and the second nucleotidesequence hybridize to one another. Note that step 4 can be performedunder reverse directional force F2, e.g., reversal of the voltagerelative to that of steps 1 and 2, so that polymerase 2250 need not bedisposed against first side 2201 of nanopore 2200.

At step 5 illustrated in FIG. 22E, directional force F1 again can beapplied, which can cause translocation of tail region 2212 in adirection away from first side 2201 of nanopore 220 and translocation ofpolymerase 2250 towards second side 2202 of nanopore 2200. For example,a voltage across nanopore 2200 again can be reversed, e.g., using asystem such as described herein with reference to FIGS. 2A-2C. However,application of force F1 at step 5 may not necessarily cause polymerase2250 to come to rest on or adjacent to first side 2201 of nanopore 2200in a manner such as illustrated in FIG. 22B. Instead, application offorce F1 (pulling towards trans) can cause a duplex defined by theinteraction (e.g., binding or hybridization) between moiety 2214 and2232 to come to rest on or adjacent to constriction 2204.Illustratively, the composition can be included in a system thatincludes measurement circuitry configured to measure a current or fluxthrough constriction 2204. During step 5, the current or flux can bebased on first moiety 2232, e.g., based upon the particular sequence ofmoiety 2232, and first nucleotide 2230 can be identifiable based uponthe current or flux. For example, moiety 2232 of first nucleotide 2230can have a different sequence than does moiety 2232′ of secondnucleotide 2230′, and can bind to a different portion (moiety) ofelongated body 2213 of tether 2210. Illustratively, the elongated tagscan include any suitable polynucleotide sequence that facilitatesdistinguishing from one another nucleotides to which such tags areattached, e.g., such as described herein with reference to FIGS.19A-21D.

At step 6 illustrated in FIG. 22F, under continued application ofdirectional force F1, after a stochastic time the duplex between moiety2214 of tether 2210 and moiety 2232 of elongated tag 2231 of nucleotide2230 dissociates in a manner analogous to that described in Derringtonet al., PNAS 2010, cited elsewhere herein. Following such dissociation,directional force F1 can cause polymerase 2250 to come to rest on oradjacent to first side 2201 of nanopore 2200 in a manner such asillustrated in FIG. 22B.

Note that other configurations suitably can be used. For example,alternatively to steps 5 and 6 respectively illustrated in FIGS. 22E and22F, elongated tag 2231 instead can be sufficiently short that theduplex between moiety 2214 of tether 2210 and moiety 2232 of elongatedtag 2231 of nucleotide 2230 does not reach the constriction underapplication of directional force F1, and instead polymerase 2250 comesto rest on or adjacent to first side 2201 of nanopore 2200 in a mannersuch as illustrated in FIG. 22B. In such embodiments, the elongated tags2231, 2231′ attached to different nucleotides 2230, 2230′ that can bebound by polymerase 2250 can include moieties 2232, 2232′ that aredifferent sequences or lengths than one another and thus interactdifferently with, e.g., hybridize differently with, moiety 2214 oftether 2210 than one another so as to cause different changes in thelength of tether 2214 in a manner analogous to that described hereinwith reference to FIGS. 7A-14. The corresponding nucleotides 2230, 2230′can be identified based on changes in current or flux based on thelength of tether 2210 caused by interactions between moiety 2214 and thecorresponding moiety 2232, 2232′. Steps 4-6 analogous to thoseillustrated in FIGS. 22D-22F can be repeated, therefore applyingAC-voltage preserving the electrodes. In yet another embodiment, theelongated tag or the elongated body can include a reporter region suchas provided elsewhere herein, and the current or flux through aperture2203 can be based on the reporter region being disposed within theaperture, and nucleotide 2230 can be identifiable based on the currentor flux.

Additionally, note that should a dysfunctional polymerase be captured,one can reverse the voltage to a very high voltage so that the captureDNA comes off and a new polymerase can be captured (repeating steps1-3).

Voltage, current, or optical waveforms can be measured for variousstates of a tether that passes through a nanopore. The voltage, current,or optical waveforms can be useful for determining results of ananalytical method carried out on a nanopore system. For example, thewaveforms can be fit to data to increase accuracy of sequencing reads.

FIGS. 24A through 24C shows three potential states for a tether thatsimulate states experienced in a nucleic acid sequencing method setforth herein. The resulting optical or electrical signals, e.g., voltagewaveforms, are shown in FIG. 24D. A protein-DNA tether conjugate iscaptured in an MspA nanopore and locked into place using a trans-sidelock oligonucleotide. An oligonucleotide complementary to a region ofthe DNA tether is then added to the cis side. Voltage is cycled between120 mV and −60 mV with approximately a 200 msec period. FIG. 24A showsthe conjugate upon the application of forward voltage and the resultingsignal is indicated at 102 of FIG. 24D. FIG. 24B shows the conjugateupon the application of the negative voltage and the resulting signal isindicated at 2400 of FIG. 24D. FIG. 24C shows hybridization of anoligonucleotide conjugate that is pulled up to the pore constriction.The duplex signal is seen prior to stripping at 2401 of FIG. 24D. Afterstripping, the system returns to the state shown in FIG. 24A while thevoltage is still at 120 mV, resulting in signal 2402.

In embodiments such as those described above with reference to FIGS.20A-22F and 24A-24D, note that the moiety of the elongated tag on thenucleotide is designed to interact with the moiety of the elongated bodyof the tether. For example, the elongated tag of the nucleotide and theelongated body of tether can include polynucleotides that hybridize(anneal) with one another, e.g., can include DNA. In such embodiments,the elongated tag of the nucleotide can include nucleotide analogs thatsubstantially do not interact with the polymerase. Discriminationbetween nucleotides can be achieved by using four different moietiesthat anneal at slightly different locations within the tether sequence.For example, in one illustrative embodiment, the 3-4 nucleotidesadjacent to the nascent duplex create maximally different blockadecurrents or fluxes corresponding to the four different nucleotides. Ifthe duplex is present, the tether can stall adjacent to theconstriction, in a manner analogous to that illustrated in FIG. 18B,20B, or 21B, for a period of time, e.g., for a few milliseconds, as theduplex is being dissociated (stripped) and a current or flux blockadereading proportional to the 3-4 nucleotides adjacent to the duplexregion is recorded. Upon stripping, the AC voltage resets the duplex viaits stochastic interaction with the DNA tag on the labeled nucleotidelocked into a tertiary closed state complex. The frequency of the ACvoltage can be tuned such that for a given AC cycle, there is arelatively low probability of detecting a diffusive event (freenucleotides), and a relatively high probability of detecting anincorporation event (nucleotide bound in a closed tertiary structure).Moreover, the number of AC cycles per nucleotide incorporation event canbe on the order of 5× to 100× oversampling to adequately distinguishbetween incorporation vs. diffusive events. Note that in such a mode ofinteraction, instead of relying on the intrinsic off-rate of theelongated tag of the nucleotide from the tether, the moieties of theelongated tag and the tether interact with one another and then can beactively stripped apart under AC voltage control, in a manner analogousto that illustrated in FIG. 18C, 20C, or 21C. The frequency of the ACvoltage (i.e. ˜100-200 Hz) can tuned to be just long enough to detectbinding of “mM concentration” tags, but significantly shorter than thedwell time of incorporation. This active stripping of the duplex canremove dependency on the exponential distribution of the off-rate.

As noted elsewhere herein, a variety of compositions suitably can beincluded in the elongated tag of the nucleotide or the elongated body ofthe tether, or both. Such compositions can include DNA, PNA, LNA, RNA,morpholinos, PEG (polyethylene glycol), and the like, and can have anysuitable length. An oligonucleotide label including an appropriatelymodified nucleotide suitably can be linked to such differentcompositions, for example, using click chemistry compatible precursorsare ideal. In one example, the nucleotide is azide-labeled, which wouldfacilitate the use of alkyne-labeled oligonucleotides which are easilysynthesized. Exemplary molecules include tetraphosphate-labelednucleotides such as shown below, in which (A) corresponds toAzide-P₄O₁₃-dTTP, and (B) corresponds to Alkyne-P₄O₁₃-dTTP. Thesenucleotides can be modified with any desired tag by using standard clickchemistry:

References on making and labeling tetraphosphate nucleotides include thefollowing, the entire contents of each of which are incorporated byreference herein:

-   Kumar, S., A. Sood, J. Wegener, P. J. Finn, S. Nampalli, J. R.    Nelson, A. Sekher, P. Mitsis, J. Macklin and C. W. Fuller, “Terminal    phosphate labeled nucleotides: synthesis, applications, and linker    effect on incorporation by DNA polymerases,” Nucleosides Nucleotides    Nucleic Acids 24(5-7): 401-408 (2005);-   Sood, A., S. Kumar, S. Nampalli, J. R. Nelson, J. Macklin and C. W.    Fuller, “Terminal phosphate-labeled nucleotides with improved    substrate properties for homogeneous nucleic acid assays,” J Am Chem    Soc 127(8): 2394-2395 (2005);-   Kumar, S., C. Tao, M. Chien, B. Hellner, A. Balijepalli, J. W.    Robertson, Z. Li, J. J. Russo, J. E. Reiner, J. J. Kasianowicz    and J. Ju, “PEG-labeled nucleotides and nanopore detection for    single molecule DNA sequencing by synthesis,” Sci Rep 2: 684 (8    pages) (2012);-   Bonnac, L., S. E. Lee, G. T. Giuffredi, L. M. Elphick, A. A.    Anderson, E. S. Child, D. J. Mann and V. Gouverneur, “Synthesis and    O-phosphorylation of 3,3,4,4-tetrafluoroaryl-C-nucleoside    analogues,” Org Biomol Chem 8(6): 1445-1454 (2010); and-   Lee, S. E., L. M. Elphick, A. A. Anderson, L. Bonnac, E. S.    Child, D. J. Mann and V. Gouverneur, “Synthesis and reactivity of    novel gamma-phosphate modified ATP analogues,” Bioorg Med Chem Lett    19(14): 3804-3807 (2009).

As noted elsewhere herein, any suitable detection method or system canbe used to detect action of a polymerase upon a nucleotide. For example,fluorescent resonance energy transfer (FRET) is an optical-baseddetection method that suitably can be used with the present compositionsso as to detect action of a polymerase upon a nucleotide. In anexemplary method, the first elongated tag of the first nucleotidefurther includes a first fluorescent resonant energy transfer (FRET)pair partner, such as a FRET acceptor or donor, and the tether furtherincludes a second FRET pair partner, such as a FRET donor or acceptor.The first FRET pair partner and the second FRET pair partner caninteract with one another responsive to the polymerase acting upon thefirst nucleotide. The method further can include detecting a firstwavelength emitted responsive to the interaction between the first FRETpair partner and the second FRET pair partner. The method further caninclude providing a second nucleotide including a second elongated tag,the second elongated tag including a third fluorescent resonant energytransfer (FRET) pair partner, e.g., a FRET acceptor or donor. The thirdFRET pair partner and the second FRET pair partner can interact with oneanother responsive to the polymerase acting upon the second nucleotide.The method further can include detecting a second wavelength emittedresponsive to the interaction between the third FRET pair partner andthe second FRET pair partner. The first and second nucleotides areindividually distinguishable from one another based on the first andsecond wavelengths.

As one illustrative example, FIG. 23A schematically illustratesexemplary nucleotides including elongated tags including respectivereporter regions and moieties that can bond to an exemplary tetherduring use in detecting action of a polymerase upon the nucleotides,according to some embodiments of the present invention. Each differenttype of nucleotide can include a corresponding elongated tag that isattached to its gamma phosphate in a manner analogous to that describedabove with reference to FIGS. 19B-19C, and that includes a correspondingfluorescent resonant energy transfer (FRET) pair partner, e.g., a FRETacceptor or donor, that can be used as a reporter region. For example,FIG. 23A schematically illustrates exemplary nucleotides 2330 includingelongated tags 2331 that include respective FRET acceptor-based reporterregions 2334 and optionally also include moieties 2332 that can bond toan exemplary tether during use in detecting action of a nucleotide by apolymerase disposed adjacent to a nanopore. As shown in FIG. 23A, A, T,C, and G nucleotides can include respectively elongated tags thatinclude different FRET acceptor-based reporter regions than one another,e.g., as respectively represented by the triangle, diamond, square, andcircle. Alternatively, the A, T, C, and G nucleotides can includerespectively elongated tags that include different FRET donor-basedreporter regions than one another, e.g., as respectively represented bythe triangle, diamond, square, and circle. For further information aboutFRET pair partners, e.g., acceptors and donors, and systems and methodsfor detecting emissions from interactions between FRET pair partners,see US Patent Publication No. 2014/0087474 to Huber and PCT PatentPublication No. WO 2014/066902 to Huber et al., the entire contents ofboth of which are incorporated by reference herein.

Optionally, elongated tags 2331 of nucleotides 2330 include moietiesthat can be suitably selected so as to hybridize to a correspondingmoiety of the permanent tether as described in greater detail below withreference to FIGS. 23B-23C. However, optional moieties 2332 of thenucleotides need not be different than one another, and indeed can bethe same as one another because the nucleotides can be distinguishablefrom one another based on differences between their FRET pairpartner-based respective reporter regions. In one illustrativeembodiment, optional moieties 2315 and 2332 have lengths of 5 to 8nucleotides. It is expected that the melting temperatures of duplexesbetween moieties on incorporating nucleotides and moieties on the tetherto be significantly more stable than an otherwise identical pair offreely diffusing oligonucleotides because the tether and theincorporating nucleotide are held in relatively fixed position relativeto another, causing an effective increase in the local concentrations ofthe moieties, as discussed above with reference to FIGS. 8A-14.Exemplary oligonucleotides that can be used as moieties are describedfurther herein, e.g., with reference to FIGS. 19A-19C.

In one example, FIG. 23B schematically illustrates a compositionincluding a tether anchored to or adjacent to a nanopore and configuredfor use in detecting action of a polymerase upon a first nucleotidebased on an interaction between the tether and a reporter region of anucleotide, according to some embodiments of the present invention. Thetether can include a FRET pair partner that interacts with therespective FRET pair partners of nucleotides 2331. In one illustrativeembodiment, the FRET pair partner of the tether is a FRET donor, and therespective FRET pair partners of the nucleotides are FRET acceptors. Inanother illustrative embodiment, the FRET pair partner of the tether isa FRET acceptor, and the respective FRET pair partners of thenucleotides are FRET donors.

The nanopore includes biological pore 2305, which can be disposed in abarrier (not specifically illustrated), e.g., a membrane of biologicalorigin such as a lipid bilayer, or a solid state membrane. Biologicalpore 2305 includes aperture 2303 and constriction 2304. The permanenttether includes head region 2311, elongated body 2313, optional moiety2315, and FRET pair partner 2316, e.g., donor or acceptor, optionallywhich can be located at or adjacent to tail region 2312 of the permanenttether. Polymerase 2350 is disposed adjacent to, and in contact with,biological pore 2305, and optionally can be anchored to biological pore2305 via a physical or chemical linkage (e.g., using click chemistry ora cysteine-maleimide bond). Polymerase 2350 is configured to receive atemplate polynucleotide 2370, e.g., circular or linear ssDNA to besequenced, to synthesize a polynucleotide 2360 having a complementarysequence to that of the ssDNA by sequentially receiving, binding, andadding nucleotides to the polynucleotide in accordance with the sequenceof the ssDNA. Head region 2311 of the tether can be anchored to anysuitable portion of biological pore 2305 that places FRET pair partner2316 sufficiently close to polymerase 2350 so as to interact with FRETpair partner-based reporter regions 2334 of nucleotides 2330 that can bebound by polymerase 2350. For example, based upon FRET donor 2316 andFRET acceptor-based reporter region 2334 being within approximately 70Angstroms of one another, a characteristic wavelength of light can beemitted based upon which nucleotide 2330 can be identified. In anotherexample, based upon FRET acceptor 2316 and FRET donor-based reporterregion 2334 being within approximately 70 Angstroms of one another, acharacteristic wavelength of light can be emitted based upon whichnucleotide 2330 can be identified.

For example, FIG. 23C schematically illustrates a detectable interactionbetween one of the reporter regions of FIG. 23A with the tether of FIG.23B during action of a polymerase upon a first nucleotide, according tosome embodiments of the present invention. As illustrated in FIG. 23C,nucleotide 2330 can include an elongated tag 2331 including first FRETpair partner-based reporter region 2334 that interacts with second FRETpair partner 2316 of the tether. In certain embodiments in which thepermanent tether includes moiety 2315 and elongated tag 2331 of thenucleotide includes moiety 2332, moieties 2315 and 2332 can interactwith one another.

In some embodiments, first FRET pair partner-based reporter region 2334and second FRET pair partner 2316 interact with one another responsiveto polymerase 2350 acting upon nucleotide 2330, and a first wavelengthemitted responsive to the interaction between first FRET partner-basedreporter region 2334 and second FRET pair partner 2316 can bedetectable. For example, as illustrated in FIG. 23C, an exemplarynucleotide 2330, e.g., T, includes an elongated tag 2331 including FRETacceptor-based reporter region 2334 and optionally oligonucleotidemoiety 2332 that can be attached to the gamma phosphate of thenucleotide 2330, e.g., via a delta phosphate linkage. Interactionbetween FRET acceptor-based reporter region 2334 and FRET donor 2316 ofthe tether causes light of a selected wavelength “λ_(T)” to be emitted.A suitable measurement circuit such as described further above withreference to FIG. 2A can be used to detect wavelength “λ_(T),” basedupon which nucleotide 2330 can be identified, e.g., as T. Optionally, inone illustrative embodiment, moiety 2315 includes a firstoligonucleotide, and moiety 2332 includes a second oligonucleotide thatis complementary to the first oligonucleotide, e.g., that hybridizes tothe first oligonucleotide. The hybridization of the secondoligonucleotide to the first oligonucleotide can cause FRETacceptor-based reporter region 2334 to become disposed adjacent to FRETdonor 2316. The action of polymerase 2350 upon nucleotide 2330 can beindividually detected based on emission of light having wavelength“λ_(T)” responsive to the interaction between FRET acceptor-basedreporter region 2334 and FRET donor 2316. It should be understood thatregion 2334 instead can be based on a FRET donor and region 2316 insteadcan be based on a FRET acceptor.

Additionally, as illustrated in FIG. 23A, each different type ofnucleotide can include a corresponding elongated tag 2331 that includesa corresponding FRET pair partner-based reporter region 2334. Each suchreporter region 2334 can be configured such that interaction betweenthat reporter region and FRET pair partner 2316 emits a correspondingwavelength based upon which the corresponding nucleotide can beidentified. For example, interaction of the FRET pair partner-basedreporter region 2334 attached to the A nucleotide 2330 (represented witha triangle) with FRET pair partner 2316 can cause emission of lighthaving wavelength “λ_(A)”; interaction of the FRET pair partner-basedreporter region 2334 attached to the C nucleotide 2330 (represented witha square) with FRET pair partner 2316 can cause emission of light havingwavelength “λ_(C)”; and interaction of the FRET pair partner-basedreporter region 2334 attached to the G nucleotide 2330 (represented witha circle) with FRET pair partner 2316 can cause emission of light havingwavelength “λ_(G)”. Accordingly, the interaction of each such reporterregion 2334 with FRET pair partner 2316 can facilitate identification ofthe corresponding nucleotide. For example, a first FRET pair partner ofa first nucleotide and the second FRET pair partner of the tether caninteract with one another responsive to the polymerase acting upon thefirst nucleotide. A first wavelength emitted responsive to theinteraction between the first FRET pair partner and the second FRET pairpartner can be detected. A second nucleotide can include a secondelongated tag including a third FRET pair partner. The third FRET pairpartner and the second FRET pair partner of the tether can interact withone another responsive to the polymerase acting upon the secondnucleotide. An optical detection system can be configured to detect asecond wavelength emitted responsive to the interaction between thethird FRET pair partner and the second FRET pair partner. The first andsecond nucleotides can be individually distinguishable from one anotherbased on the first and second wavelengths.

Elongated tags 2331 of nucleotides 2330 can be cleaved followingincorporation of such nucleotides into polynucleotide 2360 in a mannersuch as described elsewhere herein.

Additionally, note that the roles of FRET donor and acceptor suitablycan be interchanged. For example, the permanent tether can include aFRET acceptor, and the elongated tag 2331 of nucleotides 2330 caninclude FRET donor-based reporter regions 2334. Or, for example, thepermanent tether can include a FRET donor, and the elongated tag 2331 ofnucleotides 2330 can include FRET acceptor-based reporter regions 2334.Interactions between such FRET pair partners can cause light to beemitted based upon which each corresponding nucleotide can beidentified.

Exemplary Modification of Statistical Distribution of Signals

It should be noted that the dissociation of a duplex such as may beformed based on an interaction between a first moiety of an elongatedbody and a second moiety of an elongated tag responsive to an appliedvoltage can be characterized as defining a first pathway that ischaracterized by two or more kinetic constants. Additionally, the actionof a polymerase upon a nucleotide, e.g., a conformational change of thepolymerase, release of pyrophosphate, or release of the elongated tag ofthe nucleotide, can be characterized as defining a second pathway thatis characterized by two or more kinetic constants. The statisticaldistribution of signals measured (e.g., optically or electricallymeasured) during the course of obtaining measurements of the firstpathway or the second pathway can be based on the relative values ofthese kinetic constants corresponding to that pathway. For example,based upon a given kinetic constant for the first pathway or for thesecond pathway being significantly greater than other kinetic constantsfor that pathway, the kinetics of that pathway can be dominated by thatgiven kinetic constant, and the resulting statistical distribution ofsignals can be described by an exponential function. In comparison, twoor more of the kinetic constants for the first pathway or for the secondpathway can be selected so as to be of the same order of magnitude asone another, or even so as to be substantially the same as one another(e.g., to differ from one another by a factor of five or less, or fouror less, or three or less, or two or less), such that the kinetics ofthat pathway not dominated by either kinetic constant, and the resultingstatistical distribution of signals can be described by a gammafunction, in which there is substantially no probability of zero-time orvery short events that are substantially non-observable. In comparison,with an exponential distribution, there is a high probability of veryshort or zero-time events that are substantially non-observable.

One or more of the kinetic constants of the first or second pathway canbe modified in any suitable manner so as to be of the same order as oneor more other of the kinetic constants of that pathway, or even so as tobe substantially the same as one or more other of the kinetic constantsof that pathway. For example, the polymerase of any of the compositionsprovided herein can be modified so as to delay release of pyrophosphateresponsive to incorporation of a nucleotide into the first nucleotide,thus modifying at least one kinetic constant of the second pathway. Forexample, in some embodiments, the polymerase can include a modifiedrecombinant Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1,PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase. In someembodiments, the polymerase can include a modified recombinant Φ29 DNApolymerase having at least one amino acid substitution or combination ofsubstitutions selected from the group consisting of: an amino acidsubstitution at position 484, an amino acid substitution at position198, and an amino acid substitution at position 381. In someembodiments, the polymerase can include a modified recombinant Φ29 DNApolymerase having at least one amino acid substitution or combination ofsubstitutions selected from the group consisting of E375Y, K512Y, T368F,A484E, A484Y, N387L, T372Q, T372L, K478Y, 1370W, F198W, and L381A. Forfurther details regarding exemplary modified polymerases that can delayrelease of pyrophosphate responsive to incorporation of a nucleotideinto a polynucleotide, see U.S. Pat. No. 8,133,672 to Bjornson et al.,the entire contents of which are incorporated by reference herein.

As another example, one or more of the kinetic constants of the firstpathway can be modified by including along any of the present tethers asecond moiety that hybridizes with the first moiety so as to form ahairpin structure. The first and second moieties of the tether can beconfigured to dehybridize from one another in a two-step processresponsive to a voltage applied across the nanopore. An exemplarytetraphosphate modified nucleotide with a label configured to form ahairpin structure is shown in FIG. 26A. Upon hybridization with thetether, a hairpin is formed as shown in FIG. 26C. This hairpin can beexpected to have two stripping rate constants, k1 and k2, that are shownin FIG. 26C. These rate constants can be designed to be of a similarmagnitude as one another, so that when added together, they can form agamma distribution. A second exemplary tetraphosphate modifiednucleotide with two labels is shown in FIG. 26B. There are two labels,each configured to interact with the tether, as shown in FIG. 26D. Eachlabel has its own stripping rate constant, k1 and k2 respectively, andthe sum of these two rate constants for the entire stripping event canyield a gamma function. Note that any suitable phosphate moieties can beused, e.g., moieties including three, four, or six phosphates.

Illustratively, in some embodiments, a composition includes a nanoporeincluding a first side, a second side, and an aperture extending throughthe first and second sides; and a permanent tether including a headregion, a tail region, and an elongated body disposed therebetween, thehead region being anchored to or adjacent to the first side or secondside of the nanopore, and the elongated body including a reporter regionbeing movable within the aperture responsive to a first event occurringadjacent to the first side of the nanopore. Exemplary embodiments ofsuch compositions are provided above with reference at least to FIGS.1C, 1D, 1E-1M, 5A-5B, 6C-6D, 7A-7B, 8A-8B, 9A-9B, 10A-10C, 11A-11D,12A-12C, 13A-13E, 20A-20E, 22A-22E, 23A-23C, and 24A-24D.

In some embodiments, such a composition further includes a polymerasedisposed on the first side, the head region being anchored to thepolymerase. The composition further can include a first nucleotide andfirst and second polynucleotides each in contact with the polymerase,the polymerase configured to add the first nucleotide to the firstpolynucleotide based on a sequence of the second polynucleotide. Thepolymerase optionally can be modified so as to delay release ofpyrophosphate responsive to addition of the first nucleotide to thefirst polynucleotide. For example, the polymerase can include a modifiedrecombinant Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1,PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase. For example,the polymerase can include a modified recombinant Φ29 DNA polymerasehaving at least one amino acid substitution or combination ofsubstitutions selected from the group consisting of: an amino acidsubstitution at position 484, an amino acid substitution at position198, and an amino acid substitution at position 381. Or, for example,the polymerase can include a modified recombinant Φ29 DNA polymerasehaving at least one amino acid substitution or combination ofsubstitutions selected from the group consisting of E375Y, K512Y, T368F,A484E, A484Y, N387L, T372Q, T372L, K478Y, 1370W, F198W, and L381A.

Illustratively, in some embodiments, a method can include providing ananopore including a first side, a second side, and an apertureextending through the first and second sides; providing a permanenttether including a head region, a tail region, and an elongated bodydisposed therebetween, the head region being anchored to or adjacent tothe first or second side of the nanopore, the elongated body including areporter region; and moving the reporter within the aperture responsiveto a first event occurring adjacent to the first side of the nanopore.Exemplary embodiments of such methods are provided above with referenceat least to FIGS. 3A, 4A-4B, and 15.

In some embodiments, the method further can include disposing apolymerase on the first side, the head region being anchored to thepolymerase. The method further can include contacting the polymerasewith a first nucleotide and with first and second polynucleotides, thepolymerase adding the first nucleotide to the first polynucleotide basedon a sequence of the second polynucleotide. The polymerase optionallycan be modified so as to delay release of pyrophosphate responsive toaddition of the first nucleotide to the first polynucleotide. Forexample, the polymerase can include a modified recombinant Φ29, B103,GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7,PR4, PR5, PR722, or L17 polymerase. For example, the polymerase caninclude a modified recombinant Φ29 DNA polymerase having at least oneamino acid substitution or combination of substitutions selected fromthe group consisting of: an amino acid substitution at position 484, anamino acid substitution at position 198, and an amino acid substitutionat position 381. Or, for example, the polymerase can include a modifiedrecombinant Φ29 DNA polymerase having at least one amino acidsubstitution or combination of substitutions selected from the groupconsisting of E375Y, K512Y, T368F, A484E, A484Y, N387L, T372Q, T372L,K478Y, 1370W, F198W, and L381A.

Illustratively, in some embodiments, a composition can include ananopore including a first side, a second side, and an apertureextending through the first and second sides; a permanent tetherincluding a head region, a tail region, and an elongated body disposedtherebetween, the head region being anchored to or adjacent to the firstside or second side of the nanopore, the elongated body including amoiety; a polymerase disposed adjacent to the first side of thenanopore; and a first nucleotide including a first elongated tag, thefirst elongated tag including a first moiety that interacts with themoiety of the tether responsive to the polymerase acting upon the firstnucleotide. Exemplary embodiments of such compositions are providedabove with reference at least to FIGS. 7A-7B, 8A-8B, 9A-9B, 10A-10C,11A-11D, 12A-12C, 13A-13E, 16, 17A-17B, 18A-18E, 19A-19C, 20A-20E,21A-21E, 22A-22F, 23A-23C, and 24A-24D.

In some embodiments, the composition further includes first and secondpolynucleotides in contact with the polymerase, the polymeraseconfigured to add the first nucleotide to the first polynucleotide basedon a sequence of the second polynucleotide. Optionally, the polymerasecan be modified so as to delay release of pyrophosphate responsive toaddition of the first nucleotide to the first polynucleotide. Forexample, the polymerase can include a modified recombinant Φ29, B103,GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7,PR4, PR5, PR722, or L17 polymerase. For example, the polymerase caninclude a modified recombinant Φ29 DNA polymerase having at least oneamino acid substitution or combination of substitutions selected fromthe group consisting of: an amino acid substitution at position 484, anamino acid substitution at position 198, and an amino acid substitutionat position 381. Or, for example, the polymerase can include a modifiedrecombinant Φ29 DNA polymerase having at least one amino acidsubstitution or combination of substitutions selected from the groupconsisting of E375Y, K512Y, T368F, A484E, A484Y, N387L, T372Q, T372L,K478Y, 1370W, F198W, and L381A.

Additionally, or alternatively, in some embodiments, the first moietyand the moiety of the tether are configured to hybridize with oneanother so as to form a hairpin structure. A system can include such acomposition and a voltage source configured to apply a voltage acrossthe first and second sides. Non-limiting examples of hairpin structuresare described above with reference to FIGS. 26A and 26C. Exemplarysystems are described above with reference to at least FIGS. 2A and 2C,and exemplary signals that can be produced using such systems aredescribed above with reference to at least FIGS. 2B, 14, 18E, 20E, 21E,and 24D. In some embodiments, the first moiety and the moiety of thetether are configured to dehybridize from one another responsive to thevoltage in a two-step process.

Additionally, or alternatively, in some embodiments, the first elongatedtag further includes a second moiety, the composition further includinga third moiety anchored to or adjacent to the first side or second sideof the nanopore, the second moiety and the third moiety interactingresponsive to addition of the first nucleotide to the firstpolynucleotide. A system can include such a composition and a voltagesource configured to apply a voltage across the first and second sides.Non-limiting examples of third moieties are described above withreference to FIGS. 26B and 26D. Exemplary systems are described abovewith reference to at least FIGS. 2A and 2C, and exemplary signals thatcan be produced using such systems are described above with reference toat least FIGS. 2B, 14, 18E, 20E, 21E, and 24D. In some embodiments, thefirst moiety and the moiety of the tether are configured to separatefrom one another responsive to the voltage in a first process, and thesecond moiety and the third moiety are configured to separate from oneanother responsive to the voltage in a second process.

Illustratively, in some embodiments, a method includes providing ananopore including a first side, a second side, and an apertureextending through the first and second sides; providing a permanenttether including a head region, a tail region, and an elongated bodydisposed therebetween, the head region being anchored to or adjacent tothe first side or second side of the nanopore, the elongated bodyincluding a moiety; providing a polymerase disposed adjacent to thefirst side of the nanopore; providing a first nucleotide including afirst elongated tag, the first elongated tag including a moiety; actingupon the first nucleotide with the polymerase; and interacting the firstmoiety with the moiety of the tether responsive to the polymerase actingupon the first nucleotide. Exemplary methods are described above withreference at least to FIGS. 4B and 15.

In some embodiments, the method includes disposing a polymerase on thefirst side, the head region being anchored to the polymerase. The methodfurther can include contacting the polymerase with a first nucleotideand with first and second polynucleotides, the polymerase adding thefirst nucleotide to the first polynucleotide based on a sequence of thesecond polynucleotide. The polymerase optionally can be modified so asto delay release of pyrophosphate responsive to addition of the firstnucleotide to the first polynucleotide. For example, the polymerase caninclude a modified recombinant Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf,G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17polymerase. For example, the polymerase can include a modifiedrecombinant Φ29 DNA polymerase having at least one amino acidsubstitution or combination of substitutions selected from the groupconsisting of: an amino acid substitution at position 484, an amino acidsubstitution at position 198, and an amino acid substitution at position381. For example, the polymerase can include a modified recombinant Φ29DNA polymerase having at least one amino acid substitution orcombination of substitutions selected from the group consisting ofE375Y, K512Y, T368F, A484E, A484Y, N387L, T372Q, T372L, K478Y, 1370W,F198W, and L381A.

Additionally, or alternatively, in some embodiments, the first moietyand the moiety of the tether hybridize with one another so as to form ahairpin structure. Some embodiments further include applying a voltageacross the first and second sides. The first moiety and the moiety ofthe tether can dehybridize from one another responsive to the voltage ina two-step process.

Additionally, or alternatively, in some embodiments, the first elongatedtag further can include a second moiety, the composition furtherincluding a third moiety anchored to or adjacent to the first side orsecond side of the nanopore, the second moiety and the third moietyinteracting responsive to addition of the first nucleotide to the firstpolynucleotide. Some embodiments further include applying a voltageacross the first and second sides. The first moiety and the moiety ofthe tether can separate from one another responsive to the voltage in afirst process, and the second moiety and the third moiety can separatefrom one another responsive to the voltage in a second process.

Optional Modifications for Sequencing by Synthesis

In embodiments in which a polymerase adds a first nucleotide to apolynucleotide, e.g., to a first polynucleotide that is complementary toa second polynucleotide being sequenced, as in sequencing-by-synthesis(SBS), note that the first nucleotide can be coupled to any suitablereversible terminator that inhibits the polymerase from adding a secondnucleotide to the first polynucleotide until a “deblock” step isperformed.

For example, the SBS can be performed by disposing any suitableinventive composition in a flow cell, and fluid reagents for each stepin the SBS protocol can be delivered to the flow cell. For example, inSBS, extension of a nucleic acid primer along a nucleic acid template(e.g., a target polynucleotide or amplicon thereof) is monitored todetermine the sequence of nucleotides in the template. The underlyingchemical process can include polymerization (e.g., as catalyzed by apolymerase enzyme). In a particular polymerase-based SBS embodiment,nucleotides are added to a primer (thereby extending the primer) in atemplate dependent fashion such that detection of the order and type ofnucleotides added to the primer can be used to determine the sequence ofthe template. As provided herein, the nucleotides can include tags thatfacilitate identification of those nucleotides, for example, using ananopore composition set forth herein.

Flow cells provide a convenient format for housing an array ofpolymerase-attached nanopores that are subjected to an SBS techniquethat involves repeated delivery of reagents in cycles. To initiate afirst SBS cycle, one or more labeled nucleotides can be flowedinto/through a flow cell that houses an array of polymerase-attachednanopores that have formed a complex with a template nucleic acid thatis hybridized to a sequencing primer. Those sites of an array whereprimer extension causes a labeled nucleotide to be incorporated can bedetected using compositions, systems, and methods such as providedherein. Optionally, the nucleotides can further include a reversibleterminator that terminates further primer extension once a nucleotidehas been added to a primer. For example, the nucleotide that iscontacted with a complex can include a reversible terminator moiety thatgets added to a primer in such a manner that subsequent extension cannotoccur until a deblocking agent is delivered to remove the moiety. Thus,for embodiments that use reversible termination, a deblocking reagentcan be delivered to the flow cell (before or after detection occurs).Washes can be carried out between the various delivery steps. The cyclecan then be repeated n times to extend the primer by n nucleotides,thereby detecting a sequence of length n. Exemplary SBS procedures,fluidic systems and detection system components that can be readilyadapted for use in a system of method of the present disclosure aredescribed, for example, in Bentley et al., Nature 456:53-59 (2008), WO04/018497; U.S. Pat. No. 7,057,026; WO 91/06678; WO 07/123744; U.S. Pat.No. 7,329,492; U.S. Pat. No. 7,211,414; U.S. Pat. No. 7,315,019; U.S.Pat. No. 7,405,281, and US 2008/0108082, the entire contents of each ofwhich are incorporated herein by reference.

In some embodiments, the tag can be provided on the 3′ sugar position ofthe nucleotide so that the tag can be used both to identify thenucleotide and as a reversible terminator to inhibit the polymerase fromadding a second nucleotide to the first polynucleotide until a “deblock”step is performed.

In some embodiments, a reversible terminator can be provided on the 3′sugar position of the nucleotide and a tag can be provided on the base,or vice versa, to as to enhance control over, and confidence in, ahomopolymer sequencing process. For example, in embodiments in which thereversible terminator is provided on the 3′ sugar position and the tagis provided on the base, a first “deblock” process can be performed soas to remove the reversible terminator and expose the 3′ OH, and asecond “deblock” process can be performed so as to remove the tag, withany suitable order of the first and second “deblock” processes. Forexample, the tag first can be removed, based upon which the signalassociated with such a tag no longer may be observed, and the presenceof the 3′ reversible terminator can inhibit the polymerase from adding asecond nucleotide to the first polynucleotide until a second “deblock”step is performed for that reversible terminator. In such a manner,based upon the same signal being observed in a second cycle prior to thesecond deblock step being performed, the absence of signal between thefirst and second cycles can confirm that the tag was released at the endof the first cycle and added back during the second cycle, thusincreasing confidence that the polynucleotide is a homopolymer. Or, forexample, in embodiments in which the reversible terminator is providedon the base and the tag is provided on the 3′ sugar position, thepolymerase can remove the tag upon incorporation of the nucleotide intoa polynucleotide without the need for a separate chemistry step.

Additionally, note that a deblocking agent can be delivered from thetrans side (the side of the barrier opposite that of the nucleotides) ina manner that can be controlled by the selective application of avoltage gradient across the nanopore. The deblocking agent can beexpected to have an effective concentration substantially only in thevicinity of the first side of the nanopore, and can be expected to havea low concentration as it diffuses out into the bulk where the pool ofnucleotides resides so as not to deblock the nucleotides in the bulk.Alternatively, an agent that is configured to neutralize or deactivatethe deblocking agent can be present on the first side of the nanopore.This agent can be locally depleted by transport of the deblocking agentacross the nanopore, and can be expected to neutralize or deactivate thedeblocking agent further away from the nanopore, e.g., in the bulk ofthe first side, so as to inhibit deblocking of the nucleotides in thebulk.

In particular embodiments a 3′ OH blocking group can include one or moremoieties such as disclosed in PCT Publication No. WO 2004/018497, theentire contents of which are incorporated herein by reference. Forexample, the blocking group can include azidomethyl (CH₂N₃) or allyl,and the deblocking agent can include a strong reducing agent, such asTHP (tris(hydroxypropyl)phosphine). Further examples of useful blockinggroups are described, for example, in the following references, theentire contents of each of which is incorporated by reference in itsentirety: U.S. Pat. No. 7,816,503, U.S. Pat. No. 7,771,903, U.S. PatentPublication No. 2008/0108082, U.S. Patent Publication No. 2010/00317531,PCT Publication No. WO 91/06678, PCT Publication No. WO 04/018497, andPCT Publication No. WO 07/123744.

Illustratively, in some embodiments, a composition includes a nanoporeincluding a first side, a second side, and an aperture extending throughthe first and second sides; and a permanent tether including a headregion, a tail region, and an elongated body disposed therebetween, thehead region being anchored to or adjacent to the first side or secondside of the nanopore, and the elongated body including a reporter regionbeing movable within the aperture responsive to a first event occurringadjacent to the first side of the nanopore. The composition further caninclude a polymerase disposed on the first side, the head region beinganchored to the polymerase. The composition further can include a firstnucleotide and first and second polynucleotides each in contact with thepolymerase, the polymerase configured to add the first nucleotide to thefirst polynucleotide based on a sequence of the second polynucleotide.Exemplary embodiments of such compositions are provided above withreference to at least FIGS. 1F, 1M, 5A-5B, 6C-6D, 7A-7B, 8A-8B, 9A-9B,10A-10C, 11A-11D, 12A-12C, 13A-13E, 20A-20E, 22A-22E, 23A-23C, and24A-24D.

Optionally, the first nucleotide is coupled to a reversible terminatorthat inhibits the polymerase from adding a second nucleotide to thefirst polynucleotide, optionally in a manner that is controlled by theselective application of a voltage gradient across the nanopore. Thedeblocking agent can be expected to have an effective concentrationsubstantially only in the vicinity of the first side of the nanopore,and can be expected to have a low concentration as it diffuses out intothe bulk where the pool of nucleotides resides. Alternatively, an agentthat is configured to neutralize or deactivate the deblocking agent canbe present on the first side of the nanopore. This agent can be locallydepleted by transport of the deblocking agent across the nanopore, andcan be expected to neutralize or deactivate the deblocking agent furtheraway from the nanopore, e.g., in the bulk of the first side, so as toinhibit deblocking of the nucleotides in the bulk. In some embodiments,the reversible terminator is cleavable by exposure to light or heat. Forexample, the reversible terminator can be cleavable by absorption ofheat from the light. In one nonlimiting example, the reversibleterminator can include a gold nanoparticle that is sufficiently heatedby the light as to cleave the reversible terminator. Or, for example,the reversible terminator can be cleavable by a photochemical reactioninduced by the light. Or, for example, the reversible terminator can becleavable by reaction with a chemical agent. The composition further caninclude a source of the chemical agent. In some embodiments, thereversible terminator is disposed on the first side, and the source ofthe chemical agent is disposed on the second side such that the chemicalagent moves from the second side to the first side through the aperture.In one nonlimiting example, the reversible terminator includesazidomethyl (CH₂N₃), and the chemical agent includes THP.

In some embodiments, an apparatus includes any of such compositions, thecomposition is present in a flow cell, and the flow cell is configuredto replenish reagents that are in contact with the polymerase.

Illustratively, in some embodiments, a method can include providing ananopore including a first side, a second side, and an apertureextending through the first and second sides; providing a permanenttether including a head region, a tail region, and an elongated bodydisposed therebetween, the head region being anchored to or adjacent tothe first or second side of the nanopore, the elongated body including areporter region; and moving the reporter within the aperture responsiveto a first event occurring adjacent to the first side of the nanopore. Apolymerase can be disposed on the first side, the head region beinganchored to the polymerase. The method further can include contactingthe polymerase with a first nucleotide and with first and secondpolynucleotides, the polymerase adding the first nucleotide to the firstpolynucleotide based on a sequence of the second polynucleotide.Exemplary embodiments of such methods are provided above with referenceat least to FIG. 15.

Optionally, the first nucleotide can be coupled to a reversibleterminator, and the method further can include inhibiting, by thereversible terminator, the polymerase from adding a second nucleotide tothe first polynucleotide. In some embodiments, the method can includecleaving the reversible terminator by exposure to light or heat. Forexample, the method can include cleaving the reversible terminator byabsorption of heat from the light. Or, for example, the method caninclude cleaving the reversible terminator by a photochemical reactioninduced by the light. Or, for example, the method can include cleavingthe reversible terminator by reaction with a chemical agent. The methodoptionally can include providing a source of the chemical agent. Themethod optionally can include flowing fluid past the polymerase toremove the chemical agent. The method optionally can include supplyingnew reagents to the polymerase by fluid flow. In some embodiments, thereversible terminator is disposed on the first side and the source ofthe chemical agent is disposed on the second side, and the methodincludes moving the chemical agent from the second side to the firstside through the aperture. In one nonlimiting example, the reversibleterminator includes azidomethyl (CH₂N₃), and the chemical agent includesTHP.

Illustratively, in some embodiments, a composition can include ananopore including a first side, a second side, and an apertureextending through the first and second sides; a permanent tetherincluding a head region, a tail region, and an elongated body disposedtherebetween, the head region being anchored to or adjacent to the firstside or second side of the nanopore, the elongated body including amoiety; a polymerase disposed adjacent to the first side of thenanopore; and a first nucleotide including a first elongated tag, thefirst elongated tag including a first moiety that interacts with themoiety of the tether responsive to the polymerase acting upon the firstnucleotide. The composition also can include first and secondpolynucleotides in contact with the polymerase, the polymeraseconfigured to add the first nucleotide to the first polynucleotide basedon a sequence of the second polynucleotide. Exemplary embodiments ofsuch compositions are provided above with reference at least to FIGS.7A-7B, 8A-8B, 9A-9B, 10A-10C, 11A-11D, 12A-12C, 13A-13E, 16, 17A-17B,18A-18E, 19A-19C, 20A-20E, 21A-21E, 22A-22F, 23A-23C, and 24A-24D.

Optionally, the first elongated tag further can be a moiety of areversible terminator that inhibits the polymerase from adding a secondnucleotide to the first polynucleotide. For example, the reversibleterminator can be cleavable to remove the elongated tag from thepolymerase-nucleic acid complex. The cleavage can be, for example, byexposure to light or heat. For example, the reversible terminator can becleavable by absorption of heat from the light. Or, for example, thereversible terminator can be cleavable by a photochemical reactioninduced by the light. Or, for example, the reversible terminator can becleavable by reaction with a chemical agent. In some embodiments, thecomposition further includes a source of the chemical agent. In someembodiments, the reversible terminator is disposed on the first side,and the source of the chemical agent is disposed on the second side suchthat the chemical agent moves from the second side to the first sidethrough the aperture. In one nonlimiting example, the reversibleterminator includes azidomethyl (CH₂N₃), and the chemical agent includesTHP.

In some embodiments, an apparatus includes any of such compositions, thecomposition is present in a flow cell, and the flow cell is configuredto replenish reagents that are in contact with the polymerase.

Illustratively, in some embodiments, a method includes providing ananopore including a first side, a second side, and an apertureextending through the first and second sides; providing a permanenttether including a head region, a tail region, and an elongated bodydisposed therebetween, the head region being anchored to or adjacent tothe first side or second side of the nanopore, the elongated bodyincluding a moiety; providing a polymerase disposed adjacent to thefirst side of the nanopore; providing a first nucleotide including afirst elongated tag, the first elongated tag including a moiety; actingupon the first nucleotide with the polymerase; and interacting the firstmoiety with the moiety of the tether responsive to the polymerase actingupon the first nucleotide. The method further can include disposing apolymerase on the first side, the head region being anchored to thepolymerase. The method further can include contacting the polymerasewith a first nucleotide and with first and second polynucleotides, thepolymerase adding the first nucleotide to the first polynucleotide basedon a sequence of the second polynucleotide. Exemplary methods aredescribed above with reference at least to FIG. 15.

Optionally, the first elongated tag can include a reversible terminator,and the method further can include inhibiting, by the reversibleterminator, the polymerase from adding a second nucleotide to the firstpolynucleotide. For example, the method can include cleaving thereversible terminator by exposure to light or heat. For example, themethod can include cleaving the reversible terminator by absorption ofheat from the light. Or, for example, the method can include cleavingthe reversible terminator by a photochemical reaction induced by thelight. Or, for example, the method can include cleaving the reversibleterminator by reaction with a chemical agent. The method also caninclude providing a source of the chemical agent. In some embodiments,the reversible terminator is disposed on the first side and the sourceof the chemical agent is disposed on the second side, the methodincluding moving the chemical agent from the second side to the firstside through the aperture. In one nonlimiting example, the reversibleterminator includes azidomethyl (CH₂N₃), and the chemical agent includesTHP. In some embodiments, the method includes flowing fluid past thepolymerase to remove the chemical agent. The method also can includesupplying new reagents to the polymerase by fluid flow.

Other Alternative Embodiments

While various illustrative embodiments of the invention are describedabove, it will be apparent to one skilled in the art that variouschanges and modifications may be made therein without departing from theinvention. For example, although certain compositions, systems, andmethods are discussed above with reference to detecting eventsassociated with sequencing polynucleotides such as DNA or RNA, it shouldbe understood that the present compositions, systems, and methodssuitably can be adapted for use in detecting any type of event, e.g.,the motion of a molecule, or a portion thereof, that can be linked tothe presence or motion of a reporter region adjacent to a constrictionof a nanopore. The appended claims are intended to cover all suchchanges and modifications that fall within the true spirit and scope ofthe invention.

1. A composition including: a nanopore including a first side, a secondside, and an aperture extending through the first and second sides; anda permanent tether including a head region, a tail region, and anelongated body disposed therebetween, the head region being anchored toor adjacent to the first side or second side of the nanopore, and theelongated body including a reporter region being movable within theaperture responsive to a first event occurring adjacent to the firstside of the nanopore. 2-47. (canceled)
 48. A method including: providinga nanopore including a first side, a second side, and an apertureextending through the first and second sides; providing a permanenttether including a head region, a tail region, and an elongated bodydisposed therebetween, the head region being anchored to or adjacent tothe first or second side of the nanopore, the elongated body including areporter region; and moving the reporter within the aperture responsiveto a first event occurring adjacent to the first side of the nanopore.49-52. (canceled)
 53. The method of claim 48, wherein the head region isanchored to the first side of the nanopore.
 54. (canceled)
 55. Themethod of claim 48, wherein the reporter region is translationally movedtoward the first side of the nanopore responsive to the first event. 56.The method of claim 55, further including translationally moving thereporter region toward the second side after the first event. 57-61.(canceled)
 62. The method of claim 48, further including measuring afirst current or flux through the aperture or a first optical signalwhile the reporter region is moved responsive to the first event. 63.The method of claim 48, wherein a protein is disposed adjacent to thefirst side of the nanopore, and wherein the first event includes a firstconformational change of the protein.
 64. The method of claim 63,wherein the head region is anchored to the protein.
 65. The method ofclaim 64, wherein the first conformational change moves the head region,and the movement of the head region translationally moves the reporterregion. 66-68. (canceled)
 69. The method of claim 65, wherein theprotein includes a polymerase.
 70. The method of claim 69, wherein thefirst conformational change occurs responsive to the polymerase actingupon a first nucleotide.
 71. The method of claim 70, wherein the firstconformational change moves the head region, and the movement of thehead region translationally moves the reporter region.
 72. The method ofclaim 71, further including identifying the first nucleotide based on ameasured magnitude or time duration, or both, of a change in a currentor flux through the aperture or an optical signal responsive to thetranslational movement of the reporter region.
 73. The method of claim70, further including translationally moving the reporter regionresponsive to a second conformational change of the polymerase occurringresponsive to the polymerase acting upon a second nucleotide.
 74. Themethod of claim 73, further including identifying the first nucleotidebased on a measured magnitude or time duration, or both, of a firstchange in a current or flux through the aperture or a first opticalsignal responsive to the translational movement of the reporter regionresponsive to the first conformational change, and further includingidentifying the second nucleotide based on a measured magnitude or timeduration, or both, of a second change in the current or flux through theaperture or a second optical signal responsive to the translationalmovement of the reporter region responsive to the second conformationalchange. 75-76. (canceled)
 77. The method of claim 48, wherein apolymerase is disposed adjacent to the first side of the nanopore,wherein the first event includes the polymerase acting upon a firstnucleotide.
 78. The method of claim 77, wherein the first nucleotideincludes an elongated tag including a moiety that interacts with thetether.
 79. The method of claim 78, wherein the interaction of themoiety with the tether translationally moves the reporter region. 80.(canceled)
 81. The method of claim 77, wherein the tether includes afirst oligonucleotide.
 82. (canceled)
 83. The method of claim 81,wherein the moiety includes a second oligonucleotide that hybridizes tothe first oligonucleotide.
 84. The method of claim 83, wherein thehybridization of the second oligonucleotide to the first oligonucleotideshortens the tether by a first amount.
 85. The method of claim 84,further including identifying the first nucleotide based on a measuredmagnitude or time duration, or both, of a change in a current or fluxthrough the aperture or an optical signal responsive to the shorteningof the tether by the first amount. 86-94. (canceled)
 95. A compositionincluding: a nanopore including a first side, a second side, and anaperture extending through the first and second sides; a permanenttether including a head region, a tail region, and an elongated bodydisposed therebetween, the head region being anchored to or adjacent tothe first side or second side of the nanopore, the elongated bodyincluding a moiety; a polymerase disposed adjacent to the first side ofthe nanopore; and a first nucleotide including a first elongated tag,the first elongated tag including a first moiety that interacts with themoiety of the tether responsive to the polymerase acting upon the firstnucleotide. 96-162. (canceled)
 163. A method including: providing ananopore including a first side, a second side, and an apertureextending through the first and second sides; providing a permanenttether including a head region, a tail region, and an elongated bodydisposed therebetween, the head region being anchored to or adjacent tothe first side or second side of the nanopore, the elongated bodyincluding a moiety; providing a polymerase disposed adjacent to thefirst side of the nanopore; providing a first nucleotide including afirst elongated tag, the first elongated tag including a moiety; actingupon the first nucleotide with the polymerase; and interacting the firstmoiety with the moiety of the tether responsive to the polymerase actingupon the first nucleotide. 164-240. (canceled)
 241. A compositionincluding: a nanopore including a first side, a second side, and anaperture extending through the first and second sides; a permanenttether including a head region, a tail region, and an elongated bodydisposed therebetween, the head region being anchored to a polymerase,the elongated body including a moiety; the polymerase disposed adjacentto the first side of the nanopore; and a first nucleotide including afirst elongated tag, the first elongated tag including a first moietythat interacts with the moiety of the tether responsive to thepolymerase acting upon the first nucleotide. 242-252. (canceled)
 253. Amethod including: providing a nanopore including a first side, a secondside, and an aperture extending through the first and second sides;providing a permanent tether including a head region, a tail region, andan elongated body disposed therebetween, the head region being anchoredto a polymerase, the elongated body including a moiety; providing thepolymerase disposed adjacent to the first side of the nanopore;providing a first nucleotide including a first elongated tag, the firstelongated tag including a moiety; acting upon the first nucleotide withthe polymerase; and interacting the first moiety with the moiety of thetether responsive to the polymerase acting upon the first nucleotide.254-264. (canceled)
 265. A method of making a nanopore sequencingdevice, comprising: providing a chamber comprising a first liquid mediumseparated from a second liquid medium by a nanopore, the nanoporecomprising a first side in contact with the first liquid medium, asecond side in contact with the second liquid medium, and an apertureextending through the first and second sides; providing a polymerase tothe first liquid medium, wherein the polymerase comprises a tether, thetether comprising a head region, a tail region, and an elongated bodydisposed therebetween, the head region being anchored to the polymerase;providing a capture moiety to the second liquid medium; applying acurrent or flux through the nanopore to translocate the tail region ofthe tether through the nanopore; and binding the capture moiety to thetail region of the tether, thereby retaining the tether in the nanopore.266-276. (canceled)
 277. The method of claim 48, wherein polymerase isdisposed on the first side, the head region being anchored to thepolymerase, the method further including: further contacting thepolymerase with a first nucleotide and with first and secondpolynucleotides, the polymerase adding the first nucleotide to the firstpolynucleotide based on a sequence of the second polynucleotide, thepolymerase being modified so as to delay release of pyrophosphateresponsive to addition of the first nucleotide to the firstpolynucleotide. 278-316. (canceled)
 317. The method of claim 48, whereina polymerase is disposed on the first side, the head region beinganchored to the polymerase, the method further including: furthercontacting the polymerase with a first nucleotide and with first andsecond polynucleotides, the polymerase adding the first nucleotide tothe first polynucleotide based on a sequence of the secondpolynucleotide, the first nucleotide being coupled to a reversibleterminator, and inhibiting, by the reversible terminator, the polymerasefrom adding a second nucleotide to the first polynucleotide. 318-319.(canceled)
 320. The method of claim 319, the method further comprisingcleaving the reversible terminator. 321-322. (canceled)
 323. The methodof claim 317, the method further comprising cleaving the reversibleterminator by reaction with a chemical agent.
 324. (canceled)
 325. Themethod of claim 323, further comprising flowing fluid past thepolymerase to remove the chemical agent.
 326. The method of claim 325,further comprising supplying new reagents to the polymerase by fluidflow.
 327. The method of claim 326, wherein the reversible terminator isdisposed on the first side and wherein a source of the chemical agent isdisposed on the second side, the method comprising moving the chemicalagent from the second side to the first side through the aperture.328-350. (canceled)